Post on 13-Aug-2019
Spectro-microscopies
Jean SusiniEuropean Synchrotron Radiation Facility,BP220, F-38043 Grenoble Cedex , France
School on X-Ray Imaging TechniquesESRF, Grenoble, 5-6 February, 2007
Spectro-microscopy ?
Imagingtaking a picture
Spectroscopyspread the light
Photometrymeasure how much light
Spectro-microscopies
E
X-Ray Fluorescence(XRF)
Fixed
ΔE
E
X-Ray Absorption (XAS)
ΔE
E1, E2, …
E
Infrared Absorption (FTIR)
ΔEEnergy
Sig
nal i
nten
sity
2 - Spectroscopy: ΔE
1 - Microscopy: < Δx, Δy < 1µm
The interactions photons-matter provide several contrast mechanisms
Photoionization,Compton scattering
Electron shift to excited states, valence-band photoemission
Molecular rotation, torsion
Molecular vibrational states
Symmetricstretching
Anti-symmetricstretching
Bending
Relative energy of electromagneticradiation
Dissociation
Vibration
Rotation
X-rays
ultraviolet
visible
infrared
Relevant interactions of X-ray photons with matter
An electron in the given shell (e.g. K) is ejected from the atom by an external primary excitation x-ray photon, creating a vacancy.
The excitation energy from the inner atom istransferred to one of the outer electrons causing itto be ejected from the atom.
“Auger” Electron
Higher energy core electron fills empty electron level, and ejects an x-ray photon of fixed energy.
X-ray fluorescence photon
X-ray Absorption
Synchrotron based micro-imaging techniques
X-ray spectroscopy
X-rayDiffraction & scatteringX-ray
fluorescence
InfraredFTIR-spectroscopy
• Composition• Quantification
Trace element mapping
• Short range structure• Electronic structure
Oxidation/speciation mapping• Molecular groups & structure
High S/N for spectroscopyFunctional group mapping
• Long range structureCrystal orientation mappingStress/strain/texture mapping
Phase contrast X-ray imaging
• 2D/3D MorphologyHigh resolutionDensity mapping
Broad emission spectrumWavelength/energy tunability
Low emittanceBrightnessCoherence
Synchrotron based micro-imaging techniquesX-ray
fluorescence
X-ray spectroscopy
Penetration• Bulk info
Long working distance• Space for sample
Long depth of field• 3D imaging
X-rayDiffraction & scattering
InfraredFTIR-spectroscopy
• Composition• Quantification
Trace element mapping
• Short range structure• Electronic structure
Oxidation/speciation mapping• Molecular groups & structure
High S/N for spectroscopyFunctional group mapping
• Long range structureCrystal orientation mappingStress/strain/texture mapping
Phase contrast X-ray imaging
• 2D/3D MorphologyHigh resolutionDensity mapping
Synchrotron based hard X-ray microprobe
Photodiode
Undulator
monochromator
X-ray lens
Aperture
Sampleraster scanned
Fluorescence detector
CCDDiffraction
CCDAlignment & imaging
• Spatial resolution : 0.05-2µm • Spectral resolution : ΔE/E ~ 10-2 - 10-4
• Averaged flux : 109 – 1013photons/s/µm2
LN2 dewarfor cryo
sample stage
Transmission detector stage
BEAM DIRECTION
Fluorescence detectors
Visible light microscope mechanics
Sample scanning stages
Overview of the ID22 end-station
• KB mirror system
• Sample stage
• Video microscope
• Si(Li) 1 element
• Si(Li) 13 elements
• Diffraction camera
• Imaging camera
X-ray Fluorescence: a brief reminder
Element specific
Co-localization
Quantification
X-rayKen
ergy
continuum
ML
Photo-electroncontinuum
ML
K
KαKβ
Eexc
Energy dispersivedetector
energy
Inte
nsity
X-ray Fluorescence: a brief reminder
Element specific
Co-localization
Quantification
X-rayKen
ergy
continuum
ML
Photo-electroncontinuum
ML
K
KαKβ
90°
Hor. Linearpolarization
monochromaticbeam
High flux
• Low scattering (background)• Low detection limit• X-ray absorption spectroscopyXRF geometry on synchrotron beamline
X-ray Fluorescence: a brief reminder
Element specific
Co-localization
Quantification
High sensitivity
Chemical info.
X-rayKen
ergy
continuum
ML
Photo-electroncontinuum
ML
K
KαKβ
90°
Hor. Linearpolarization
monochromaticbeam
High flux
• Low scattering (background)• Low detection limit• X-ray Absorption spectroscopy
X-ray Lines and Transitions
J. A. Bearden, Rev. Mod. Phys. 39, 78 (1967)
Photon energies, in electron volts, of principal K-, and L- shell emission lines
From http://xdb.lbl.gov/xdb.pdf
The fluorescence yield ωω is the probability that an x-ray photon will be
emitted as a result of ionization of a specific shell. For a given series of X-ray lines (e.g., the K series), ω is numerically equal to the ratio of K photons escaping from the atom to the ratio of original K-shell ionizations.
The fluorescence yield for K lines increases monotonically as a function of atomic number, and algebraic models accurately predict the empirical data.
This factor means that the sensitivity of the x-ray method decreases for the lighter elements. The decrease, however, is partly compensated by the higher photo-absorption cross sections in light elements.
Example:• zinc (Z=30) ωk = 0.45
• sodium (Z=11) ωk = 0.02
M.O. Krause, J. Phys. Chem. Ref. Data, 89,(1979)
Atomic number
Fluo
resc
ence
yie
ld ω
Phytoextraction in hyper accumulator plants
Green and low cost strategy for soil cleaning
Requires knowledge on the mechanisms of metal accumulation
M.P. Isaure et al., Biochimie, 2006
Anthropogenic activities
Cesium (Cs)Nuclear activities
Cadmium (Cd)Industrial (mining), agricultural
activities (fertilizers)
XRF mapping in Trichomes of Arabidopsis Thaliana
P
70 µm
S Cd K Ca
Eex: 5.8 keV, probe size: 0.4x0.2μm2, dwell time: 800 ms/pixel.
M.P. Isaure et al., Biochimie, in press
Cs accumulation in Arabidopsis Thaliana
10μm
20 µm
Eex: 5.8 keV, probe size: 0.4x0.2μm2, dwell time: 800 ms/pixel.
M.P. Isaure et al., Biochimie, 2006
Fluorescence tomography (3D-µXRF)
Pixel- by-pixel acquisition 2D-Slice or 3D-VolumeSinogram(s)
2D X-rayFocusing device
EDS
Sample
Beam
Diode #1
Diode #2
Fluorescence tomography (3D-µXRF)
The reconstruction problem is far more difficult compared to transmission tomography
self absorption corrections
µ(Ea, x) is a priori unknown
weak fluorescence signal for light elements
Pixel- by-pixel acquisition 2D-Slice or 3D-VolumeSinogram(s)
Fluorescence tomography (3D-µXRF)
Pixel- by-pixel acquisition 2D-Slice or 3D-VolumeSinogram(s)
Algorithmic solution:Optimal estimation of attenuation maps by combination of transmission, fluorescence and Compton tomographies
• B. Golosio et al., J. Appl. Phys. 94(1), 145 (2003)
Geometrical solution:Collimation of the detection angle to define a voxel: confocal geometry
• B. L. Vincze et al., Anal.Chem., 76(22) (2004)
Combining several signals
Photoelectric contribution to the absorption: σphoto~Z4
Transmission tomography• Provides the absorption coefficient
distribution µ(E0, x)
Compton tomography• provides information on the electronic density spatial
distribution
Compton scattering cross section σCompton~Z
Fluorescence tomography• information on the internal spatial
distribution of specific elements
QUANTIFICATION?Integration of the information(transmission+Compton+fluorescence)
B. Golosio et al., J. Appl. Phys. 94(1), 145 (2003)
• Foraminifera
Single-cell marine animals
Fossilized calcium carbonate shells
Proxies for past oceanic conditions (climate)
200 μm
Globorotalia inflata
X-ray fluorescence tomography
P. Bleuet et al., SPIE, 2006
X-ray radiograph
Transmission Ca Fe
Cu Zn Ni
Fluo-tomography: 3D volume rendering
Foraminifera (Globorotalia inflata)
Quantification on 2D slicesTransmission
50μm 0
50
cm-1
Compton
0
0.69
g/cm-3
50μm
0.009
0
g/cm-3
0.009
0
S0.008
0
g/cm-3
Cl0.522
0
g/cm-3
Ca
0.009
0
g/cm-3
Zn0.001
0
g/cm-3
Cu
X-ray Fluorescence Tomography – “confocal geometry”
Polycapillary
Coincidingfocii
FocussedX-ray beam
XRFdetector
Spatial resolution: 5-15µm
L. Vincze et al., Anal.Chem., 76(22) (2004)
Si(Li) detector
I0
Optical microscope
90°
Focusing lensSample
CCDAlignment
& imaging
ID18F
3D-Confocal XRF for tomography
diamond KK200
inclusion #2
Information on the geochemical environment and conditions in which the diamond was formed
Chemistry at several 100km depth
• Voxel-size: 7 × 7 × 7 µm3
• Full 3D analysis: 28830 spectra (voxel)• dwell time: 3 s/voxel
Y, Zr Sr
3D-rendering based on measured Sr, Y and Zr-Kα distributions
L. Vincze et al., Anal.Chem., 76(22) (2004)F.E. Brenker et al., EPSL, 236, (2005)
From 3D-confocal XRF to quantification (Vekemans et al. JAAS, 2004)
Element Phase 1 Phase 2Ca 29.6 % 18.6 %Sr 431 ppm 48 ppmY 9 ppm 61 ppmZr 35 ppm 233 ppm
Zr,Y rich phaseWalstromite structured CaSiO3
Sr-rich phaseLamite Ca2SiO4
depth Z
horizontal X
verti
cal Y
Sr, Th, Y/Zr composed image
unusual high Ca concentration
“existence of a Ca-rich diamond
reservoir at depth below 300 km”
Quantification using NIST SRM 613
CaMn
Fe
PbHf Th
SrZr
Y
Phase 1 (Sr)Phase 2 (Y,Zr)
Zr
YScatter peaks
Ca: 260 ppm, Sr: 1.2 ppm, Zr: 1.1 ppm
Detection limits (LT=100 s):
X-ray Absorption Near Edge Structure (XANES)
• Local site symmetry• Oxidation state • Orbital occupancy
1s
Continuumstates
Rydbergstates
σ
σ∗
ππ∗
X-rayEnergy
Nor
m. f
luo
yiel
d or
-alo
gI/I
0
XANES:electronic transitions to bound states,
nearly bond states or continuum
Chromium compounds
N
24 electrons
- 6 electronsCr VI (hexavalent)
- 3 electronsCr III (trivalent)
Chromium in cells
0
1(a.u.)
Potassium Cr (total)
Cr(VI)
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5.96 5.98 6 6.02 6.04 6.06 6.08 6.1
CrCl3PbCrO4
Tran
smitt
ed in
tens
ity (a
.u.)
Energy (keV)
R. Ortega et al. , Chem. Res. Toxicol., 5, (2005)
Micrograph
10 µm
Micro-XANES at the Sulfur K-edge in Pinna Nobilis
Mineral prisms
Organic matrix
0
0.2
0.4
0.6
0.8
1
2460 2470 2480 2490 2500
Energy (keV)
2.460 2.470 2.480 2.490 2.500
Energy (keV)
MethionineC-S-C
ChondroitineC-SO
4
CystineC-S-S-C
CysteineH-S-C
Y. Dauphin et al., Comparative Biochemistry and Physiology, Part A 132, (2002)
Chemical mapping of Sulphur species in Pinna Nobilis
Visible light microscope
Y. Dauphin et al., Journal of Marine Biology, 142, (2003) Y. Dauphin et al., J. Structural Biology, 132, (2003)
10µm
0.0
0.5 0.1
0.1a.u.
Fluorescence yield
• probe: 0.20x0.30 mm2
• dwell time: 2 sec/pixel
E=2473eV E=2482eV
SEM
Sulfur
Alternative strategy for fast and stable hard X-ray µ-XAS
Two-crystal fixed exit scanning monochromator+ large energy range for EXAFS+ high flux (XRF)+ mature technology- source of non-statistical noises(crystal motions, lack of beam stability, precision)
- relatively slow (10s-1s)
Wavelength (energy) dispersive polychromator+ fast (1s-1µs)+ stable- low flux- limited energy range and less flexible- fluorescence more difficult
ID24-ESRF: an energy dispersive spectrometer on undulator source
VFM1
HFM
VFM2
PLC
I0IF
VFM1
HFM
VFM2
PLC
I0IF
Slits: 50mm/s -> 1000eV/s
10-4< ΔE/E < 10-1
Tuning of flux vs ΔE/E
Scanning the slits = energy scan
1 pixel = 1 EXAFS or NEXAFS spectrum100x100pixels = 10000 spectra in 2 hrs acquisition
With an optimized optical scheme: 300x300nm2
5-15 keV5 x 5 µm2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
7100 7120 7140 7160 7180A
bsor
ptio
n (a
.u.)
Energy (eV)
S. Pascarelli et al., J. of Synchrotron Radiation, 2006
Redox and speciation mapping of metamorphic rocks
Sambagawa (Japan)
P, T conditions of formation of rocks
Thermodynamical models depend critically on oxidation state of Fe
Need for quantitative map of Fe redox at the µm level
S. Pascarelli et al., J. of Synchrotron Radiation, 2006
Redox and speciation mapping of metamorphic rocks
Sambagawa (Japan)
y (µ
m)
50
100
150
50 100 150 200 250 300 350
x (µm)
• Pixel size: 4x4µm2
• 3000spectra (1.5hrs)
0
1
2
7120 7140 7160 7180
Nom
aliz
ed a
bsor
banc
e
Energy (eV)
Chlorite Fe(II)Chlorite Fe(III)
Average
Single pixel
a)
From spectra to speciation mapping ?3 basic criteria:• Edge jump: Fe content• Edge position: Fe oxidation state• Absorbance at a defined energy: Fe speciation
Hei
ght
(µm
)
50
100
150
50 100 150 200 250 300 350Length (µm)
Length (µm)
Hei
ght
(µm
)
50
100
150 0.1
0.2
0.3
0.4
50 100 150 200 250 300 350Length (µm)
7121
7123
7125
7126
7122
7124
50 100 150 200 250 300 350Length (µm)
50
100
150Hei
ght
(µm
)
M. Muñoz et al., G-cubed, (2006)
Edge jumpFe content
Edge positionFe oxidation state
0.0
0.5
1.0
1.5
2.0
2.5
7100 7120 7140 7160 7180
Abs
orpt
ion
(a.u
.)
Energy (eV)
Chlorite Fe2+
Chlorite Fe3+
Phengite
Quartz
single pixel XANES
50 100 150 200 250 300 350Length (µm)
Hei
ght
(µm
)
50
100
150 0.750.800.850.900.95
Absorbance at a defined energyFe speciation
Quartz
Chlor 2+
Chlor 3+ Phengite
Blackening of Pompeian Cinnabar paintings
Red = cinnabar (HgS)
• M. Cotte, J. Susini, ESRF, Grenoble, France
• A. Moscato & C. Gratziu, Università di Pisa, Pisa,Italy
• A. Bertagnini, Istituto Nazionale di Geofisica e Vulcanologia, Pisa, Italy
• M. Pagano, Soprintendenza per i Beni Archeologici del Molise, Campobasso, Italy
Blackening of Pompeian Cinnabar paintings
Red = cinnabar (HgS)
• Excavation started in 1988 and was completed in 1992
• Rapid blackening since 1990
“Villa Sora”, in Torre del Greco near by Pompei
Blackening of Pompeian Cinnabar paintings
no alteration low alteration high alteration
• Cinnabar β−cinnabar?• Role of the lime substrate (CaCO3)?• Superficial or deep alteration?• Role of sulfur?• Other elements (e.g. cera punica) ?
X-ray fluorescence and elemental mapping
Energy (keV)
Cou
nts
SHg
AlMg
Na
SiCl
Compton
K
Rayleigh
1.2 1.6 2.0 4.03.63.22.82.4
104
103
102
101
Raw dataFit
(PyMca by A. Sole, ESRF)
Composition maps by X-ray Fluorescence
Hg Na
Si KAl
min
maxCl
S
S
ClHg Na
Si KAl
2mm
Semi-quantification?N
orm
aliz
ed fl
uore
scen
ce y
ield
(a.u
.)
Energy (keV)
2.46 2.47 2.48 2.49 2.50 2.51
Nor
mal
ized
fluo
resc
ence
yie
ld (a
.u.)
Energy (keV)
2.49 2.51 2.532.45 2.47
Sulfur K-edge XANES
XANES Sulfur K-edge
2.46 2.47 2.48 2.49 2.50
Energy (keV)
Nor
mal
ized
fluo
resc
ence
yie
ld (a
.u.)
HgS (cinnabar)
HgS (metacinnabar)
As2S3
Sb2S3
red Cd2S3
yellow Cd2S3
S8
Hg3S2Cl2
2.46 2.47 2.48 2.49 2.50 2.51 2.52N
orm
aliz
ed fl
uore
scen
ce y
ield
(a.u
.)
Energy (keV)
CaSO4.2H2O
CuSO4.H2O
PbSO4
FeSO4.nH2O
Na3Ca(Al3Si3O12)S
Al2(SO4)3
Sulfides Sulfates
Semi-quantification?N
orm
aliz
ed fl
uore
scen
ce y
ield
(a.u
.)
Energy (keV)
2.46 2.47 2.48 2.49 2.50 2.51
Nor
mal
ized
fluo
resc
ence
yie
ld (a
.u.)
2.45 2.47 2.49 2.51 2.53
Energy (keV)
Fit
Semi-quantification: sulfur compounds
gypsumcorderoite
sulfurcinnabar
Proportion (%)
0 20 40 60 80 100
(n=8)
(n=10)
(n=9)
(n=9)
(n=10)
(n=6)
Nor
mal
ized
fluo
resc
ence
yie
ld (a
.u.)
Energy (keV)
2.46 2.47 2.48 2.49 2.50 2.51
Semi-quantification: Chlorine compounds
2.81 2.82 2.83 2.84 2.85 2.86 2.87
Nor
mal
ized
fluo
resc
ence
yie
ld (a
.u.)
Energy (keV)
calomel
corderoite
terlinguaite
NaCl+KCl+CaCl2
Proportion (%)
0 20 40 60 80 100
(n=5)
(n=11)
(n=9)
(n=7)
(n=10)
Chemical mapping?
Nor
mal
ized
fluo
resc
ence
yie
ld (a
.u.)
Energy (keV)
2.49 2.51 2.532.45 2.47
[Sulfides] ∝I(2.471) - I(2.460)
I(2.510) - I(2.460)
[Sulfates] ∝I(2.482) - I(2.510)
I(2.510) - I(2.460)
2.471 2.482 2.5102.460
∝[re
duce
d su
lfur]
∝[o
xidi
zed
sulfu
r]
Chemical mapping: reduced vs oxidised Sulfur
min
max
Reduced sulfur Oxidised sulfurLight microscopy
SummaryAttributes of multi-keV X-Ray spectro-microscopies
X-ray Fluorescence
Micro-spectroscopy (XANES)
Larger focal lengths (> 20mm) Larger depth of focus (> 100µm)
• Space for sample environment• 3D imaging
Higher penetrationPhase contrast
• Chemical state specificity
• Microscopy on thick samples• Lower radiation damage (?)
• Trace element detection & mapping• Quantitative fluorescence analysis
Multi-technique approach• Micro-Fluorescence• Micro-diffraction• 3D imaging• Spectroscopies
In-situ experimentscontrolled sample environment
A common scientific case : trace element analysis in heterogeneous systems
Biology & MedicineEarth & Planetary Sciences
Environmental Sciences
Oxidation states
Quantification Co-localisationTrace elements
monitoringRedox reactions
2D/3D mappingXRF
XANES
Infrared spectroscopy
Asymmetric stretch2350 cm-1
Vertical bend666 cm-1
Symmetric stretch(not IR active)
Horizontal benddegenerate mode same
motion as above but rotated by 90o
CO2O = C = O
An IR active mode must involve a change in the dipole moment of the molecule = charge imbalance in the molecule
absorption =-log (It /Io)Io It
Infrared spectroscopy
Asymmetric stretch2350 cm-1
Vertical bend666 cm-1
Symmetric stretch(not IR active)
Horizontal benddegenerate mode same
motion as above but rotated by 90o
CO2O = C = O
Each functional group has an ensemble of motions ( vibrational) specificof the molecular group (fingerprint)
These motions ( or vibrational frequencies) are detected under« resonant » excitation in the energy domain 0.495 eV-0.062eV or 2.5 to 20µm or 4000-500 cm-1
There are databanks of spectra, which allow a rapid search and identification.
Infrared spectroscopy: some figures…
Vibration frequencies (and wave-numbers) are inversely proportional to atomic masses
• C-H stretch (3000 cm-1)• C-O stretch (1000-1300 cm-1)• C-Br stretch (600 cm-1)
Vibration frequencies (and wave-numbers) are proportional to bond strength
• C-C stretch (1000 cm-1)• C=C stretch (1600 cm-1)• C≡C stretch (2200 cm-1)
Near-IR: 12500 – 4000 cm-1
Mid-IR: 4000 – 400 cm-1
Far-IR: 400 – 10 cm-1
v
µ-w
0.8 to 2.5 µm
2.5 to 25 µm
25 to 100 µm
1 eV ~ 8100 cm-1
An example of spectrum (biological sample)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Abs
orba
nce
1000 1500 2000 2500 3000 3500 4000
Wavenumbers (cm-1)
Amide IC=O
Amide IIC-N and N-H
CH2 and CH3stretching CH3 bending
Broad O-H and N-HStretching
PO2- stretching
C-O stretch
CO2
PO32- stretching of
phosphorylated proteins
Synchrotron infrared radiation: Two modes of emission
Bending magnet emission:
Edge emission:@ 10 µm
Synchrotron Infrared microscopy
Edge radiation
transfer line
spectrometermicroscope Beam
conditioning
Synchrotron source: brightness advantage
10000 1000 100 10 110 8
10 9
1010
1011
1012
1013
1014
1015
1016
1017
1018
1019
Practical limits
Universal Equation
2000K Black Body
Synchrotron Radiation
Phot
ons/
sec/
0.1%
bw/m
m2 /s
tr2
Wavelength (microns)BRIGHTNESS
Spatial Resolution
Signal-to-Noise
Data Collection
BROADBAND
Spectroscopy
Spectro-microscopy
Chemical mapping
A confocal microscope
Two confocal Schwarzschild objectives:
• focus the light onto the sample
• collect the light and relay it to the detector.
Diffraction-limited resolution of λ/2 ( λ: 2→12µm)
G.L. Carr, Rev. of Scientific Instruments, 2001, 72, 1613
Synchrotron vs Globar
0
5
10
15
20
0 10 20 30 40
Aperture (µm)P
eak
to p
eak
inte
nsity
(V)
Synchrotron
Globar
6×6 µm2 22×22 µm2
Synchrotron Globar
0102030405060708090
100
5 7 9 11 13Aperture (µm)
Iinte
nsity
ratio
ESRF-ID21
Synchrotron FTIR Microscopy
P. Dumas et al. Vibr. Spectr. 32 (2003).
500s
16s
0.0 1.0 (a.u.)
30 µm
cuticle
C. Merigoux et al.,Biochimica & Biophysica Acta, 1619, 53, (2003)
Ca - XRF0.2x0.2µm2
Infrared Spectra
Various calcium sites in human hair shaft
1000 1500 2000 2500 3000 3500 Wavenumbers (cm-1)
CH3-(CH2)n-XO
C NH
0.0 1.0 (a.u.)
30 µm
cuticle
C. Merigoux et al.,Biochimica & Biophysica Acta, 1619, 53, (2003)
Various calcium sites in human hair shaft
Two different « types » of lipids in cuticule and medulla
Ca - XRF0.2x0.2µm2
Protein distribution in cortex
Outlook
Multimodal analysisMultivariate data processing
Quantity Quality%
ppmppb structural
chemicalelemental
Scale
mm
µm
nm
3DIn-situ experiments
FluxDetection technology
Source and Optics