Post on 25-May-2020
Basics in steady state and time resolved spectroscopy
Dr Christian Litwinski
Group Prof Nyokong
Synthesis of Phthalocyanines and NanoparticlesSensorsElectrochemistry
Photodynamic TherapyNonlinear Optical Materials
3
Contentbull Steady state absorption spectroscopy
Absorbance purity extinction coefficient ε aggregation
bull Steady state fluorescence spectroscopyFluorescence Investigation of FRET
bull Time correlated single photon counting (TCSPC)Lifetime of first excited state Investigation of FRET
bull Laser flash photolysis
Population and lifetime of triplet state
bull Singlet oxygen luminescence detectionSinglet oxygen quantum yield
bull X-ray diffraction spectroscopyStructure and size of samples
bull X-ray photoelectron spectroscopy
elemental composition chemical state and electronic state of the elements in a material
4
Electromagnetic radiation
light
identification
origin
radio waves
sun
nuclear
magnetic
resonance
electronsmol
rot
mol
vibr
outer
atomic
shell
inner
atomic
shell
atomic
core
beta-
tronsynchrotron
cosmic
radiation
spin change NMRESR
spectroscopy
Rotation and
vibration
IR
spectroscopy
Valence electron
transitions
UVVIS
spectroscopy
Core electron
transition crystal
structure
X-ray spectroscopy
3 D Crystal
structure
X-ray
tomography
Elemental
particles
Accelerator
Jabłoński Diagram
5
S0
S2
S1
T1
T2-1
5s)
Flu
ore
scen
ce(1
0-9s
10 -8
s)
(10
-12s
10 -1
1s)
10 -8s
10 -6s10 0s
Ph
osp
ho
resc
ence
(10
-6s
10 0s)
T-T Absorption
T3
S0
S2
S1
T1
T2A
bso
rpti
on
(10
-15
(10-9
s1
0 -8
s)
(10
-12s
10 -1
1s)
10 -8s
10 -6s10 0s
(10
-6s
10 0s)
S0
S2
S1
T1
T2-
(10-9
s1
0 -8
s)
Inte
rnal
Co
nv
ersi
on
(10
-12s
10 -1
1s)
Intersystemcrossing
10 -8s
10 -6s10 0s
(10
-6s
10 0s)
Ab
sorp
tio
n
T3
N
NH
N
HN
N
NH
N
HN
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
NN
N
N N
N
N N
PorphinPhthalocyanin
Naphthalocyanin
Tetrabenzo-Porphyrin
Tetraaza-Porphyrin
Tetrapyrazino-tetraazaporphyrin
N
H N
NH
N
NN
HN
N
N
Anthracocyanin
Phthalocyanine
7
Steady State Absorption
Absorption spectrumAbsorption spectrum shows the fraction of incident light absorbed by the
material over a range of frequencies
Lamp
Mirror
MCMirrorMirror Mirror
Mirror
Detector
Sample
Reference
Amplifier
Aperture
Computer
ε = molar extinction coefficient [ε] =M-1cm-1
c = concentration [c] = mol
σ = absorption cross section of one mol [σ] = cm2
s = pathway [s] = cm
n = number of mol
Lambert-Beer-Law
8
picture by University of Bremen
I(s)= I0-IA
I = I0 10(-εcs) or I = I0 e(-σns)
OD
Determinaton of ε
I = I0 10(-εcs)
0000002 0000004 0000006 0000008 0000010
01
02
03
04
05
06
07
08
ε (845nm) = 75491 M-1 cm
-1
OD
CS
DPCH2 in Toluene
OD= -εcs
N
NH
N
NN
HN
N
N
R R
R
R
R R
N
NH
N
NN
HN
N
NR
R
R R
R R
300 400 500 600 700 800 900
00
02
04
06
08
10
Ab
so
rption
[a
u]
Wellenlaumlnge [nm]
H2Pc-H
2Pc
ZnPc-ZnPc
Origin of spectra in Pcs
e g
a 2 u
b 2 u
a 1 u
e g
b 1 u
b 2 u
x xx xx xx x
x x
a 2 u
B1
B2
Q
Ring Metallated Pc (D4h)
10
Q
B
Metallated Pc (D4h)
11
Also N L and C bands at high
energy in transparent
solvents chloroform
dichloromethane
eg split -hence Q band split
N
NN
N
NN
N
N
H
H H2Pc
Unmetallated Pc complexes (D2h)
Metallated Pc (D4h)
eg
a1u
a2u
Q bands
12
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Group Prof Nyokong
Synthesis of Phthalocyanines and NanoparticlesSensorsElectrochemistry
Photodynamic TherapyNonlinear Optical Materials
3
Contentbull Steady state absorption spectroscopy
Absorbance purity extinction coefficient ε aggregation
bull Steady state fluorescence spectroscopyFluorescence Investigation of FRET
bull Time correlated single photon counting (TCSPC)Lifetime of first excited state Investigation of FRET
bull Laser flash photolysis
Population and lifetime of triplet state
bull Singlet oxygen luminescence detectionSinglet oxygen quantum yield
bull X-ray diffraction spectroscopyStructure and size of samples
bull X-ray photoelectron spectroscopy
elemental composition chemical state and electronic state of the elements in a material
4
Electromagnetic radiation
light
identification
origin
radio waves
sun
nuclear
magnetic
resonance
electronsmol
rot
mol
vibr
outer
atomic
shell
inner
atomic
shell
atomic
core
beta-
tronsynchrotron
cosmic
radiation
spin change NMRESR
spectroscopy
Rotation and
vibration
IR
spectroscopy
Valence electron
transitions
UVVIS
spectroscopy
Core electron
transition crystal
structure
X-ray spectroscopy
3 D Crystal
structure
X-ray
tomography
Elemental
particles
Accelerator
Jabłoński Diagram
5
S0
S2
S1
T1
T2-1
5s)
Flu
ore
scen
ce(1
0-9s
10 -8
s)
(10
-12s
10 -1
1s)
10 -8s
10 -6s10 0s
Ph
osp
ho
resc
ence
(10
-6s
10 0s)
T-T Absorption
T3
S0
S2
S1
T1
T2A
bso
rpti
on
(10
-15
(10-9
s1
0 -8
s)
(10
-12s
10 -1
1s)
10 -8s
10 -6s10 0s
(10
-6s
10 0s)
S0
S2
S1
T1
T2-
(10-9
s1
0 -8
s)
Inte
rnal
Co
nv
ersi
on
(10
-12s
10 -1
1s)
Intersystemcrossing
10 -8s
10 -6s10 0s
(10
-6s
10 0s)
Ab
sorp
tio
n
T3
N
NH
N
HN
N
NH
N
HN
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
NN
N
N N
N
N N
PorphinPhthalocyanin
Naphthalocyanin
Tetrabenzo-Porphyrin
Tetraaza-Porphyrin
Tetrapyrazino-tetraazaporphyrin
N
H N
NH
N
NN
HN
N
N
Anthracocyanin
Phthalocyanine
7
Steady State Absorption
Absorption spectrumAbsorption spectrum shows the fraction of incident light absorbed by the
material over a range of frequencies
Lamp
Mirror
MCMirrorMirror Mirror
Mirror
Detector
Sample
Reference
Amplifier
Aperture
Computer
ε = molar extinction coefficient [ε] =M-1cm-1
c = concentration [c] = mol
σ = absorption cross section of one mol [σ] = cm2
s = pathway [s] = cm
n = number of mol
Lambert-Beer-Law
8
picture by University of Bremen
I(s)= I0-IA
I = I0 10(-εcs) or I = I0 e(-σns)
OD
Determinaton of ε
I = I0 10(-εcs)
0000002 0000004 0000006 0000008 0000010
01
02
03
04
05
06
07
08
ε (845nm) = 75491 M-1 cm
-1
OD
CS
DPCH2 in Toluene
OD= -εcs
N
NH
N
NN
HN
N
N
R R
R
R
R R
N
NH
N
NN
HN
N
NR
R
R R
R R
300 400 500 600 700 800 900
00
02
04
06
08
10
Ab
so
rption
[a
u]
Wellenlaumlnge [nm]
H2Pc-H
2Pc
ZnPc-ZnPc
Origin of spectra in Pcs
e g
a 2 u
b 2 u
a 1 u
e g
b 1 u
b 2 u
x xx xx xx x
x x
a 2 u
B1
B2
Q
Ring Metallated Pc (D4h)
10
Q
B
Metallated Pc (D4h)
11
Also N L and C bands at high
energy in transparent
solvents chloroform
dichloromethane
eg split -hence Q band split
N
NN
N
NN
N
N
H
H H2Pc
Unmetallated Pc complexes (D2h)
Metallated Pc (D4h)
eg
a1u
a2u
Q bands
12
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
3
Contentbull Steady state absorption spectroscopy
Absorbance purity extinction coefficient ε aggregation
bull Steady state fluorescence spectroscopyFluorescence Investigation of FRET
bull Time correlated single photon counting (TCSPC)Lifetime of first excited state Investigation of FRET
bull Laser flash photolysis
Population and lifetime of triplet state
bull Singlet oxygen luminescence detectionSinglet oxygen quantum yield
bull X-ray diffraction spectroscopyStructure and size of samples
bull X-ray photoelectron spectroscopy
elemental composition chemical state and electronic state of the elements in a material
4
Electromagnetic radiation
light
identification
origin
radio waves
sun
nuclear
magnetic
resonance
electronsmol
rot
mol
vibr
outer
atomic
shell
inner
atomic
shell
atomic
core
beta-
tronsynchrotron
cosmic
radiation
spin change NMRESR
spectroscopy
Rotation and
vibration
IR
spectroscopy
Valence electron
transitions
UVVIS
spectroscopy
Core electron
transition crystal
structure
X-ray spectroscopy
3 D Crystal
structure
X-ray
tomography
Elemental
particles
Accelerator
Jabłoński Diagram
5
S0
S2
S1
T1
T2-1
5s)
Flu
ore
scen
ce(1
0-9s
10 -8
s)
(10
-12s
10 -1
1s)
10 -8s
10 -6s10 0s
Ph
osp
ho
resc
ence
(10
-6s
10 0s)
T-T Absorption
T3
S0
S2
S1
T1
T2A
bso
rpti
on
(10
-15
(10-9
s1
0 -8
s)
(10
-12s
10 -1
1s)
10 -8s
10 -6s10 0s
(10
-6s
10 0s)
S0
S2
S1
T1
T2-
(10-9
s1
0 -8
s)
Inte
rnal
Co
nv
ersi
on
(10
-12s
10 -1
1s)
Intersystemcrossing
10 -8s
10 -6s10 0s
(10
-6s
10 0s)
Ab
sorp
tio
n
T3
N
NH
N
HN
N
NH
N
HN
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
NN
N
N N
N
N N
PorphinPhthalocyanin
Naphthalocyanin
Tetrabenzo-Porphyrin
Tetraaza-Porphyrin
Tetrapyrazino-tetraazaporphyrin
N
H N
NH
N
NN
HN
N
N
Anthracocyanin
Phthalocyanine
7
Steady State Absorption
Absorption spectrumAbsorption spectrum shows the fraction of incident light absorbed by the
material over a range of frequencies
Lamp
Mirror
MCMirrorMirror Mirror
Mirror
Detector
Sample
Reference
Amplifier
Aperture
Computer
ε = molar extinction coefficient [ε] =M-1cm-1
c = concentration [c] = mol
σ = absorption cross section of one mol [σ] = cm2
s = pathway [s] = cm
n = number of mol
Lambert-Beer-Law
8
picture by University of Bremen
I(s)= I0-IA
I = I0 10(-εcs) or I = I0 e(-σns)
OD
Determinaton of ε
I = I0 10(-εcs)
0000002 0000004 0000006 0000008 0000010
01
02
03
04
05
06
07
08
ε (845nm) = 75491 M-1 cm
-1
OD
CS
DPCH2 in Toluene
OD= -εcs
N
NH
N
NN
HN
N
N
R R
R
R
R R
N
NH
N
NN
HN
N
NR
R
R R
R R
300 400 500 600 700 800 900
00
02
04
06
08
10
Ab
so
rption
[a
u]
Wellenlaumlnge [nm]
H2Pc-H
2Pc
ZnPc-ZnPc
Origin of spectra in Pcs
e g
a 2 u
b 2 u
a 1 u
e g
b 1 u
b 2 u
x xx xx xx x
x x
a 2 u
B1
B2
Q
Ring Metallated Pc (D4h)
10
Q
B
Metallated Pc (D4h)
11
Also N L and C bands at high
energy in transparent
solvents chloroform
dichloromethane
eg split -hence Q band split
N
NN
N
NN
N
N
H
H H2Pc
Unmetallated Pc complexes (D2h)
Metallated Pc (D4h)
eg
a1u
a2u
Q bands
12
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
4
Electromagnetic radiation
light
identification
origin
radio waves
sun
nuclear
magnetic
resonance
electronsmol
rot
mol
vibr
outer
atomic
shell
inner
atomic
shell
atomic
core
beta-
tronsynchrotron
cosmic
radiation
spin change NMRESR
spectroscopy
Rotation and
vibration
IR
spectroscopy
Valence electron
transitions
UVVIS
spectroscopy
Core electron
transition crystal
structure
X-ray spectroscopy
3 D Crystal
structure
X-ray
tomography
Elemental
particles
Accelerator
Jabłoński Diagram
5
S0
S2
S1
T1
T2-1
5s)
Flu
ore
scen
ce(1
0-9s
10 -8
s)
(10
-12s
10 -1
1s)
10 -8s
10 -6s10 0s
Ph
osp
ho
resc
ence
(10
-6s
10 0s)
T-T Absorption
T3
S0
S2
S1
T1
T2A
bso
rpti
on
(10
-15
(10-9
s1
0 -8
s)
(10
-12s
10 -1
1s)
10 -8s
10 -6s10 0s
(10
-6s
10 0s)
S0
S2
S1
T1
T2-
(10-9
s1
0 -8
s)
Inte
rnal
Co
nv
ersi
on
(10
-12s
10 -1
1s)
Intersystemcrossing
10 -8s
10 -6s10 0s
(10
-6s
10 0s)
Ab
sorp
tio
n
T3
N
NH
N
HN
N
NH
N
HN
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
NN
N
N N
N
N N
PorphinPhthalocyanin
Naphthalocyanin
Tetrabenzo-Porphyrin
Tetraaza-Porphyrin
Tetrapyrazino-tetraazaporphyrin
N
H N
NH
N
NN
HN
N
N
Anthracocyanin
Phthalocyanine
7
Steady State Absorption
Absorption spectrumAbsorption spectrum shows the fraction of incident light absorbed by the
material over a range of frequencies
Lamp
Mirror
MCMirrorMirror Mirror
Mirror
Detector
Sample
Reference
Amplifier
Aperture
Computer
ε = molar extinction coefficient [ε] =M-1cm-1
c = concentration [c] = mol
σ = absorption cross section of one mol [σ] = cm2
s = pathway [s] = cm
n = number of mol
Lambert-Beer-Law
8
picture by University of Bremen
I(s)= I0-IA
I = I0 10(-εcs) or I = I0 e(-σns)
OD
Determinaton of ε
I = I0 10(-εcs)
0000002 0000004 0000006 0000008 0000010
01
02
03
04
05
06
07
08
ε (845nm) = 75491 M-1 cm
-1
OD
CS
DPCH2 in Toluene
OD= -εcs
N
NH
N
NN
HN
N
N
R R
R
R
R R
N
NH
N
NN
HN
N
NR
R
R R
R R
300 400 500 600 700 800 900
00
02
04
06
08
10
Ab
so
rption
[a
u]
Wellenlaumlnge [nm]
H2Pc-H
2Pc
ZnPc-ZnPc
Origin of spectra in Pcs
e g
a 2 u
b 2 u
a 1 u
e g
b 1 u
b 2 u
x xx xx xx x
x x
a 2 u
B1
B2
Q
Ring Metallated Pc (D4h)
10
Q
B
Metallated Pc (D4h)
11
Also N L and C bands at high
energy in transparent
solvents chloroform
dichloromethane
eg split -hence Q band split
N
NN
N
NN
N
N
H
H H2Pc
Unmetallated Pc complexes (D2h)
Metallated Pc (D4h)
eg
a1u
a2u
Q bands
12
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Jabłoński Diagram
5
S0
S2
S1
T1
T2-1
5s)
Flu
ore
scen
ce(1
0-9s
10 -8
s)
(10
-12s
10 -1
1s)
10 -8s
10 -6s10 0s
Ph
osp
ho
resc
ence
(10
-6s
10 0s)
T-T Absorption
T3
S0
S2
S1
T1
T2A
bso
rpti
on
(10
-15
(10-9
s1
0 -8
s)
(10
-12s
10 -1
1s)
10 -8s
10 -6s10 0s
(10
-6s
10 0s)
S0
S2
S1
T1
T2-
(10-9
s1
0 -8
s)
Inte
rnal
Co
nv
ersi
on
(10
-12s
10 -1
1s)
Intersystemcrossing
10 -8s
10 -6s10 0s
(10
-6s
10 0s)
Ab
sorp
tio
n
T3
N
NH
N
HN
N
NH
N
HN
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
NN
N
N N
N
N N
PorphinPhthalocyanin
Naphthalocyanin
Tetrabenzo-Porphyrin
Tetraaza-Porphyrin
Tetrapyrazino-tetraazaporphyrin
N
H N
NH
N
NN
HN
N
N
Anthracocyanin
Phthalocyanine
7
Steady State Absorption
Absorption spectrumAbsorption spectrum shows the fraction of incident light absorbed by the
material over a range of frequencies
Lamp
Mirror
MCMirrorMirror Mirror
Mirror
Detector
Sample
Reference
Amplifier
Aperture
Computer
ε = molar extinction coefficient [ε] =M-1cm-1
c = concentration [c] = mol
σ = absorption cross section of one mol [σ] = cm2
s = pathway [s] = cm
n = number of mol
Lambert-Beer-Law
8
picture by University of Bremen
I(s)= I0-IA
I = I0 10(-εcs) or I = I0 e(-σns)
OD
Determinaton of ε
I = I0 10(-εcs)
0000002 0000004 0000006 0000008 0000010
01
02
03
04
05
06
07
08
ε (845nm) = 75491 M-1 cm
-1
OD
CS
DPCH2 in Toluene
OD= -εcs
N
NH
N
NN
HN
N
N
R R
R
R
R R
N
NH
N
NN
HN
N
NR
R
R R
R R
300 400 500 600 700 800 900
00
02
04
06
08
10
Ab
so
rption
[a
u]
Wellenlaumlnge [nm]
H2Pc-H
2Pc
ZnPc-ZnPc
Origin of spectra in Pcs
e g
a 2 u
b 2 u
a 1 u
e g
b 1 u
b 2 u
x xx xx xx x
x x
a 2 u
B1
B2
Q
Ring Metallated Pc (D4h)
10
Q
B
Metallated Pc (D4h)
11
Also N L and C bands at high
energy in transparent
solvents chloroform
dichloromethane
eg split -hence Q band split
N
NN
N
NN
N
N
H
H H2Pc
Unmetallated Pc complexes (D2h)
Metallated Pc (D4h)
eg
a1u
a2u
Q bands
12
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
N
NH
N
HN
N
NH
N
HN
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
N
NH
N
NN
HN
N
N
NN
N
N N
N
N N
PorphinPhthalocyanin
Naphthalocyanin
Tetrabenzo-Porphyrin
Tetraaza-Porphyrin
Tetrapyrazino-tetraazaporphyrin
N
H N
NH
N
NN
HN
N
N
Anthracocyanin
Phthalocyanine
7
Steady State Absorption
Absorption spectrumAbsorption spectrum shows the fraction of incident light absorbed by the
material over a range of frequencies
Lamp
Mirror
MCMirrorMirror Mirror
Mirror
Detector
Sample
Reference
Amplifier
Aperture
Computer
ε = molar extinction coefficient [ε] =M-1cm-1
c = concentration [c] = mol
σ = absorption cross section of one mol [σ] = cm2
s = pathway [s] = cm
n = number of mol
Lambert-Beer-Law
8
picture by University of Bremen
I(s)= I0-IA
I = I0 10(-εcs) or I = I0 e(-σns)
OD
Determinaton of ε
I = I0 10(-εcs)
0000002 0000004 0000006 0000008 0000010
01
02
03
04
05
06
07
08
ε (845nm) = 75491 M-1 cm
-1
OD
CS
DPCH2 in Toluene
OD= -εcs
N
NH
N
NN
HN
N
N
R R
R
R
R R
N
NH
N
NN
HN
N
NR
R
R R
R R
300 400 500 600 700 800 900
00
02
04
06
08
10
Ab
so
rption
[a
u]
Wellenlaumlnge [nm]
H2Pc-H
2Pc
ZnPc-ZnPc
Origin of spectra in Pcs
e g
a 2 u
b 2 u
a 1 u
e g
b 1 u
b 2 u
x xx xx xx x
x x
a 2 u
B1
B2
Q
Ring Metallated Pc (D4h)
10
Q
B
Metallated Pc (D4h)
11
Also N L and C bands at high
energy in transparent
solvents chloroform
dichloromethane
eg split -hence Q band split
N
NN
N
NN
N
N
H
H H2Pc
Unmetallated Pc complexes (D2h)
Metallated Pc (D4h)
eg
a1u
a2u
Q bands
12
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
7
Steady State Absorption
Absorption spectrumAbsorption spectrum shows the fraction of incident light absorbed by the
material over a range of frequencies
Lamp
Mirror
MCMirrorMirror Mirror
Mirror
Detector
Sample
Reference
Amplifier
Aperture
Computer
ε = molar extinction coefficient [ε] =M-1cm-1
c = concentration [c] = mol
σ = absorption cross section of one mol [σ] = cm2
s = pathway [s] = cm
n = number of mol
Lambert-Beer-Law
8
picture by University of Bremen
I(s)= I0-IA
I = I0 10(-εcs) or I = I0 e(-σns)
OD
Determinaton of ε
I = I0 10(-εcs)
0000002 0000004 0000006 0000008 0000010
01
02
03
04
05
06
07
08
ε (845nm) = 75491 M-1 cm
-1
OD
CS
DPCH2 in Toluene
OD= -εcs
N
NH
N
NN
HN
N
N
R R
R
R
R R
N
NH
N
NN
HN
N
NR
R
R R
R R
300 400 500 600 700 800 900
00
02
04
06
08
10
Ab
so
rption
[a
u]
Wellenlaumlnge [nm]
H2Pc-H
2Pc
ZnPc-ZnPc
Origin of spectra in Pcs
e g
a 2 u
b 2 u
a 1 u
e g
b 1 u
b 2 u
x xx xx xx x
x x
a 2 u
B1
B2
Q
Ring Metallated Pc (D4h)
10
Q
B
Metallated Pc (D4h)
11
Also N L and C bands at high
energy in transparent
solvents chloroform
dichloromethane
eg split -hence Q band split
N
NN
N
NN
N
N
H
H H2Pc
Unmetallated Pc complexes (D2h)
Metallated Pc (D4h)
eg
a1u
a2u
Q bands
12
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
ε = molar extinction coefficient [ε] =M-1cm-1
c = concentration [c] = mol
σ = absorption cross section of one mol [σ] = cm2
s = pathway [s] = cm
n = number of mol
Lambert-Beer-Law
8
picture by University of Bremen
I(s)= I0-IA
I = I0 10(-εcs) or I = I0 e(-σns)
OD
Determinaton of ε
I = I0 10(-εcs)
0000002 0000004 0000006 0000008 0000010
01
02
03
04
05
06
07
08
ε (845nm) = 75491 M-1 cm
-1
OD
CS
DPCH2 in Toluene
OD= -εcs
N
NH
N
NN
HN
N
N
R R
R
R
R R
N
NH
N
NN
HN
N
NR
R
R R
R R
300 400 500 600 700 800 900
00
02
04
06
08
10
Ab
so
rption
[a
u]
Wellenlaumlnge [nm]
H2Pc-H
2Pc
ZnPc-ZnPc
Origin of spectra in Pcs
e g
a 2 u
b 2 u
a 1 u
e g
b 1 u
b 2 u
x xx xx xx x
x x
a 2 u
B1
B2
Q
Ring Metallated Pc (D4h)
10
Q
B
Metallated Pc (D4h)
11
Also N L and C bands at high
energy in transparent
solvents chloroform
dichloromethane
eg split -hence Q band split
N
NN
N
NN
N
N
H
H H2Pc
Unmetallated Pc complexes (D2h)
Metallated Pc (D4h)
eg
a1u
a2u
Q bands
12
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Determinaton of ε
I = I0 10(-εcs)
0000002 0000004 0000006 0000008 0000010
01
02
03
04
05
06
07
08
ε (845nm) = 75491 M-1 cm
-1
OD
CS
DPCH2 in Toluene
OD= -εcs
N
NH
N
NN
HN
N
N
R R
R
R
R R
N
NH
N
NN
HN
N
NR
R
R R
R R
300 400 500 600 700 800 900
00
02
04
06
08
10
Ab
so
rption
[a
u]
Wellenlaumlnge [nm]
H2Pc-H
2Pc
ZnPc-ZnPc
Origin of spectra in Pcs
e g
a 2 u
b 2 u
a 1 u
e g
b 1 u
b 2 u
x xx xx xx x
x x
a 2 u
B1
B2
Q
Ring Metallated Pc (D4h)
10
Q
B
Metallated Pc (D4h)
11
Also N L and C bands at high
energy in transparent
solvents chloroform
dichloromethane
eg split -hence Q band split
N
NN
N
NN
N
N
H
H H2Pc
Unmetallated Pc complexes (D2h)
Metallated Pc (D4h)
eg
a1u
a2u
Q bands
12
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Origin of spectra in Pcs
e g
a 2 u
b 2 u
a 1 u
e g
b 1 u
b 2 u
x xx xx xx x
x x
a 2 u
B1
B2
Q
Ring Metallated Pc (D4h)
10
Q
B
Metallated Pc (D4h)
11
Also N L and C bands at high
energy in transparent
solvents chloroform
dichloromethane
eg split -hence Q band split
N
NN
N
NN
N
N
H
H H2Pc
Unmetallated Pc complexes (D2h)
Metallated Pc (D4h)
eg
a1u
a2u
Q bands
12
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Q
B
Metallated Pc (D4h)
11
Also N L and C bands at high
energy in transparent
solvents chloroform
dichloromethane
eg split -hence Q band split
N
NN
N
NN
N
N
H
H H2Pc
Unmetallated Pc complexes (D2h)
Metallated Pc (D4h)
eg
a1u
a2u
Q bands
12
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
eg split -hence Q band split
N
NN
N
NN
N
N
H
H H2Pc
Unmetallated Pc complexes (D2h)
Metallated Pc (D4h)
eg
a1u
a2u
Q bands
12
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
H2Pc spectra ndash Not split in basic solvents (eg DMSO
pyridine)
DMSO
DMSO
13
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
H2Pc
H2Nc
H2Ac
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
N N
N
NN
N
N
N
H
H
The Q band splitting of H2Pcs becomes smaller at
longer wavelength
300 700 1000nm
14
Expansion of the π electron system
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Q
Q+
Q-
Monomer Dimer
1Eu(1)
1A1g
1Eu(1)
1Eg(1)
J aggregates ndash edge to edge red shifted ndash NOT COMMON
H aggregates ndash face to face blue shifted - COMMON
15
Aggregation
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
0
02
04
300 500 700
Ab
sorb
an
ce
Wavelength (nm)
monomeraggregate
-H
16
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Zn
N N
N
NN
N
N
NHOOC
HOOC
HOOC COOH
COOH
COOH
COOHHOOC
0
02
04
06
08
1
500 600 700 800
Ab
so
rba
nce
Wavelength (nm)
N
N
N
N
N
N
N
N
HOOC
COOHHOOC
Si ClCl
COOH
Plurality of ligands and aggregation
Not aggregated
17
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
N
N
N
N
N
N
N
NTi
O
R1
R1
R1
R1
R2
R2
R2
R2
Effects of solvents on aggregation
Durmas Ahsen Nyokong Dalton Trans (2007)1235
SCH(CH2O(CH
2CH
2O)
2C
2H
5)
2R2 =
Chloroform
18
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Proof of non-aggregation Beerrsquos law
19
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
0
02
04
06
08
1
12
14
350 450 550 650 750 850
Ab
sorb
ance
Wavelengthnm
Proof of aggregation dimer peaks
decreases faster than monomer on
dilution
20
Proof of non-aggregation Beerrsquos law
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Proof of aggregation Effects of
Surfactants
pH 74
300 400 500 600 700 800
Ab
sorb
an
ce
Wavelength (nm)
No surfactant
surfactant
21
(eg Cremophore EL )(eg Cremophore EL )
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
22
Steady State Fluorescence
Picture by Mizower
Fluorescence spectrumFluorescence spectrum is plot of fluorescence intensity vs registration
wavelength (frequency energy) at one excitation wavelength
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
23
Steady State Fluorescence
600 700 800
00
02
04
06
08
10
Ab
sorp
tion
[au
]
Wellenlنnge [nm]
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Fluorescence1 Stokes Law a maximum of fluorescence
spectrum is red-shifted compared to a
maximum of the corresponding absorption
spectrum (Reasons Franck-Condon rule)
2 Mirror Image Rule a fluorescence
spectrum (plotted in energy scale) strongly
resembles the mirror image of the
absorption spectrum (Reason the vibrational
energy level spacing is similar for the ground
and excited states)
3 Universal Relationship (betwenn Abs-Flu)
Wfl(ν) = C(T)sdotK(ν)Abssdotexp(-hνkT)
4 Kasha-Vavilov Rule the fluorescence
spectrum shows very little dependence on
the wavelength of the excitation (Reasons
the emission occurs exclusively from the lowest
singlet excited electronic state)
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Fluorescence
Low solvent polarity
∆E1
∆E0
En
erg
y
eqS0
neqS0
FCS1 neq
S1
FCS0
eqS1
aν 0
flν t
flν infinν fl
High solvent polarity
( ) R
t
flflfl
t
fl eτ
minusinfininfin sdotνminusν+ν=ν 0
Dependency of the spectral
position of a fluorescence
band on time
τR is solvent relaxation time
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Quantum Yield
bull yield of product relative to amount of photons
absorbed
bull sum off all quantum yields in a process is
26
absorbed photons ofnumber
molecules formedproduct ofnumber =Φ
Examples
ΦR = Quantum yield of reaction
Φfl = Fluorescence quantum yield
ΦPh = Phosphorescence quantum yield
ΦISC = Intersystem crossing quantum yield
ΦIC = Internal conversion quantum yield
Φ∆ = Singlet oxygen quantum yield
1le
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Fluorescence Quantum Yield Φf
bull absolute measurements spectrometer with
Integrating sphere (Ulbricht Sphere)
bull comparative measurements optical spectrometer
27
absorbed photons ofnumber
photons cefluorescen ofnumber =Φ fl
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
inte
nsityλ
wavelength [nm]
SymPcH2
600 700 800
0
200
400
600
800
1000
1200
inte
nsity
wavelength [nm]
H2TPP
Example comparative
measurement
I=51575
Φf=011
I=330989
Φf=071
2
2
stdsamplestd
samplestdSample
stdsamplenODI
nODIΦ=Φ
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
perp
perp
+
minus=
II
IIP
||
||
Polarization
perp
perp
sdot+
minus=
II
IIr
2||
||
Anisotropy
r
rP
P
Pr
+=
minus=
2
3
3
2
5
1cos3
cos3
1cos3 2
02
2
0
minusθ=
θ+
minusθ= rP
Perrin expression
40202
1
3
100 leleminusleleminus rP
5
1cos3 2
0
minusβ= rrββββ
Polarizer
Analyzer
At ββββ = 547degdegdegdeg (Magic Angle)
r = 0
Lakowicz J Principles of fluorescence spectroscopy Plenum Press New York 1999
Polarization of Fluorescence
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
bull Dipole-dipole resonance interaction between photoexcited donor
molecule (D) and acceptor molecule (A) (in the most cases A is in the
ground state)
Energy of dipole-dipole interaction between donor and acceptor molecules
( ) ( )( )
minus= RR ADADddRR
M micromicromicromicromicromicromicromicromicromicromicromicromicromicromicromicro23
31
Foerster Resonace Energy Tranfer
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln900046
0
45
0
2
ν
ννεν
τπ
Φχsdot= int
dI
RNnk A
n
DD
a
Ddd
DA
( ) 1~~ =ννint dIn
D( )22 coscos3cos AD θθminusα=χ
bull Overlap of the fluorescence spectrum of D
and the absorption spectrum of A
bull High extinction coefficient of A molecule
bull Short distance between D and A molecules
bull Right orientation of transition dipole
moments (χχχχ2 nenenene 0)
bull High fluorescence quantum yield of D
High probability of EET if
Foerster Resonace Energy Tranfer
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
1
6
0
0
τ
=R
Rk
DDA
Rate of dipole-dipole EET
( ) ( ) ~
~~~
128
10ln9000445
0
26
0ν
ννεν
π
Φχsdot= int
dI
NnR A
n
D
a
D
If R = R0 then the fluorescence of the D is quenched by a factor 2
Foerster Resonace Energy Tranfer
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Factors influencing FRET Efficiency
Good spectral overlap
500 600 700 800
No
rma
lize
d In
ten
sit
y
Wavelength (nm)
Pc absQD ems
Pc flu
λλλελ partint= 4)()( PcQDfJ
DF
DAF
ssEffΦ
Φminus= 1
660
60
rR
REff
+=
JnRDFΦtimes= minus42236
0 1088 κ
Foumlrster radius
FRET efficiency
iiii ταΣτ =DF
DAF
trEffτ
τminus= 1
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
34
TCSPC
bull Determination of fluorescence lifetime
bullbull Time resolution 60 ps up to 1 Time resolution 60 ps up to 1 μμss
bullbull Measurements with low fluorescence quantum yield possible Measurements with low fluorescence quantum yield possible
(lt1)(lt1)
4 6 8 10 12 14 16 18 20
0
2000
4000
6000
8000
10000
Time [ns]
Co
un
ts
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
TCSPC Principle
rArr repeat the dwell time measurement many times ldquocount how many photons arrived after what timerdquoie build a histogram
rArr time axis is not continuous but divided into ldquotime binsrdquo
rArr Note not more than one photon per laser pulse can be registered
Photon emission is a stochastic process
Photon
1
laser
pulse
Photon
2
laser
pulse
eg 24 ns eg 45 ns
laser
pulse
no
photon
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
TCSPC principle
bull detection of single photons caused by a periodic light signal
bull light intensity is so low that probability to dectect one photon in one period is very low
bull thus periods with more than one photon are very rare
bull the time difference between laser pulse and every detected photon will be measured
bull with many pulses (millions) one gets a distribution of time differences which correspond to the fluorescence lifetime
bull time resolution up 20ps with a MCP-PMT and 150 ps with a PMT
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
15052008 Seminarvortrag Ch Litwinski 37
ldquoStartrdquo ldquoStop2rdquo
Laser
ldquoStop1rdquo
Luminescence
∆t
N
ldquoPile-uprdquo effect
TCSPC principle
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
38
TCSPC setup
Monochromator
Histogram
electronic
PCldquoStoprdquo
ldquoStartrdquo
Filter
Sample
(MCP)-PMT
Photo diode
Beam splitter
Laser
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
bull PMT pulses from photomultiplier
bull SYNC synchron pulses
bull CFD Constant-Fraction Discriminator
measures the exact time of detection
bull TAC Time-to-Amplitude Convertercondensator which loads up in the time between
SYNC signal and PMT signal
bull PGA Programmable Gain Amplifier
amplifies the TAC- output voltage with
tunable factor
bull ADC Analog-to-Digital-Converter
convert voltage to a number between 0
(fastest photons) and eg 4096 (latest)
bull MEM Memory
has in our case 4096 counter which will be
increased by 1 if a photon is detected in the
certain time period
TCSPC schematic diagram
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Data Analysis
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
Zaumlhlr
ate
Zeit [ns]
Rodamin 6G
Apparatefunktion
4 6 8
1
10
100
1000
10000 Apparatefunktion
Zeit [ns]
Zauml
hlra
te45 ps
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
)(exp)( ztIRFt
atpfi i
iminusotimes
= sum
τModel function
( )
2
2 )()(sum
minus=
N
i
ii
t
tdtpf
σχχ2-value
Data )(i
td
Data AnalysisData Analysis
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
2 4 6 8 10 12 14 16 18 20 22
0
2000
4000
6000
8000
10000
2 4 6 8 10 12 14 16 18 20 22
-6
-4
-2
0
2
4
6
Zaumlhlrate
Zeit [ns]
Messung
Fit
Re
sid
uie
n
Zeit [ns]
τ= 373plusmn001
Data AnalysisData Analysis
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
Example
N N
Zn
N N
N
N
N
NH
O
O
N
N N
Zn
N N
N
N
N
N
O O
O
S N
S OH
O
DCC48 hrt
QD
QD
0
50
100
150
200
250
300
350
400
500 550 600 650 700 750 800
Inte
ns
ity
(au
)
Wavelength (nm)
(a)
(b)
(c)
Compound Relative A1 ττττF-1 (ns)
(plusmnplusmnplusmnplusmn 05)
Relative A2 ττττF-2 (ns)
(plusmnplusmnplusmnplusmn 03)
CdTe MPA QD 057
(061a)
264
(263 a)
043
(039 a)
34
(43 a)
QD-ZnttbIPc-linked 021 96 079 17
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
45
Decay Associated Fluorescence
Spectroscopy (DAFS)bull Fluoresence lifetime of a homogenius species is independant from the
detection wavelength
bull Measurement of time resolved fluroescence at different detectionwavelengths
bull Data analysis at different detection wavelength is given by the model
bull Global fit over all data sets
Result Fluorescence spectra of differents species with
different fluorescence lifetimes
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
46
640 660 680 700 720 740 760 780 800
00
02
04
06
08
10
N
orm
In
tensitauml
t
Wellenlaumlnge [nm]
Fluoreszenz
DAFS
τfl
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
47
Data
=M
N
M
n
N
aa
a
aa
S
Κ
ΜΜ
Κ
1
11
1
)( ωλMatrix of amplitude
coefficients
Matrix of lifetimes
otimesminus
otimesminus
=Π
)()exp(
)()exp(
)(
1
1
N
N
l
l
nn
ttIRFt
ttIRFt
t
δτ
δτ
δτ Κ
N species
M Wavelength
L time channels
Nilanglang1
Mw langlang1
Ll langlang1
=M
L
M
l
L
II
I
II
D
1
11
1
ωω
Κ
Data AnalysisData Analysis
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
48
)()(~
nntSD δτλ Πsdot=Model matrix
22 )
~(
11ωωω
ω
σχρ
οDDLM
global minus= sumχ2-Value
Data AnalysisData Analysis
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
49
Example
M= H2 H2Pc
M= Zn ZnPc
O
CH3
CH3
R=
N
N
N
NN
N
N
N
R R
R
R
R R
M
R
R
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
M= H2 H2Pc-H2Pc
M= Zn ZnPc-ZnPc
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
50
650 700 750 800 850 900 950 1000
00
02
04
06
08
10
N
orm
F
luore
szen
z
Wellenlaumlnge [nm]
Fluoreszenz
ZnPc ZnPc-ZnPc RTZn LTZn
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
N
N
N
NN
N
N
N
M M M
N
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
N
N
N
NN
N
N
N
R
R
R R
R
R
M M
MN
N
N
NN
N
N
N
R R
R
R
R R
M
R
RN
N
N
NN
N
N
N
R R
R
R
R R
N
N
N
NN
N
N
NR
R
R R
R R
M M
ExampleExample
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
51
750 nm 790 nm
800 820 840 860 880 90000
02
04
06
08
10
12
14
16
740 nm
In
ten
sitauml
t [a
u]
Wellenlaumlnge [nm]
ZnPc-ZnPc
Anregung bei
ExampleExampleFluoreszenz
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
52
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
000
001
002
003
004
005
006
DAFS
A
mp
litud
e [au
]
Wellenlaumlnge [nm]
ExampleExample
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
53
ZnPc-ZnPc
τfl = 076 plusmn 002 ns
800 820 840 860 880 900
00
02
04
06
08
10 DAFS
Fluoreszenz
No
rm In
ten
sitaumlt
Wellenlaumlnge [nm]
ExampleExample
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
54
882 nm
860 nm
800 820 840 860 880 900 920
00
02
04
06
08
10
836 nm
N
orm
In
ten
sitauml
t
Wellenlaumlnge [nm]
Anregung bei
H2Pc-H2Pc
ExampleExampleFluoreszenz
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS
55
800 810 820 830 840 850 860 870 880 890 900 910
0000
0025
0050
τ1 = 043 plusmn 002ns
τ2 = 081 plusmn 002ns
τ3 = 087 plusmn 002 ns
A
mplit
ude
[a
u]
Wellenlaumlnge [nm]
H2Pc-H2Pc
ExampleExampleDAFS