Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan...
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Transcript of Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan...
Modular Fiber Optic System for Intramural Functional
Fluorescence MeasurementDean TaiDean Tai
Bryan CaldwellBryan CaldwellIan LeGriceIan LeGriceJohn HarveyJohn Harvey
Sally RutherfordSally RutherfordBruce SmaillBruce Smaill
Outline
Functional fluorescence imaging systemFunctional fluorescence imaging systemOptrodeOptrodeModular fiber fluorescence imaging Modular fiber fluorescence imaging
systemsystemResultsResultsFuFutureture Development Development
Functional Fluorescent Indicator
Non-ratiometric probesNon-ratiometric probesData often expressed as relative Data often expressed as relative
change in intensity (ΔF/F)change in intensity (ΔF/F)Used for relative comparisonUsed for relative comparison
From Molecular Probes Handbook From Molecular Probes Handbook Figure 19.24Figure 19.24
Ratiometric probesRatiometric probesExpressed as ratios between Expressed as ratios between
wavelengths wavelengths (eg. F(eg. F500500nm/Fnm/F400400nm)nm)
Used for absolute comparisonUsed for absolute comparison
From Molecular Probes Handbook From Molecular Probes Handbook Figure 20.4Figure 20.4
Fibre Optics Based Fluorescence Imaging System
Coherent illumination sourcesCoherent illumination sources Intramural multi-site recordingIntramural multi-site recording Higher spatial resolutionHigher spatial resolution 3D mapping3D mapping Dual-wavelength recordingDual-wavelength recording
>600nm
520~600nm
Optrode
Modular All-Fibre Imaging System
Solid state laserSolid state laser Fibre couplerFibre coupler
1-to-8 fibre splitter1-to-8 fibre splitter
Laser Noise
PPeak-to-peak noise eak-to-peak noise ≈≈ 3.78% 3.78%
RMS RMS noise noise ≈≈ 1.21% 1.21%
PPeak-to-peak noise eak-to-peak noise ≈≈ 0.19%0.19%
RMS noise RMS noise ≈≈ 0.11%0.11%
Photobleaching Rate(for voltage-sensitive dye, di-4-ANEPPS)
Photobleaching rates behave Photobleaching rates behave similarly for both similarly for both wavelengthswavelengths
Decay rate is significantly Decay rate is significantly higher with 488nm higher with 488nm excitationexcitation
Initial signal level is ~ 2:1 Initial signal level is ~ 2:1 with 488nm and 532nm with 488nm and 532nm excitation respectivelyexcitation respectively
532nm is a more suitable 532nm is a more suitable excitation source for long excitation source for long duration or repeated duration or repeated recordings at the same site recordings at the same site
Initial Signal Level
532nm
488nm
Mean (a.u.) 18.7 36.8
Standard Deviation
(a.u.)14.9 23.2
Spectral Shift Spectral response as a Spectral response as a
function of membrane function of membrane potential (-87, -65 and -potential (-87, -65 and -44mV for 4, 15 and 44mV for 4, 15 and 50mmol/L of potassium 50mmol/L of potassium concentration)concentration)
With 532nm and 488nm With 532nm and 488nm excitation sourcesexcitation sources
Quantitatively expressed Quantitatively expressed as ratio between the as ratio between the intensity of two windows intensity of two windows above and below the above and below the isosbestic pointisosbestic point
Quantifying the Spectral Shift
With 532nm With 532nm excitation, Fexcitation, F650650/F/F580580 was measuredwas measured
With 488nm With 488nm excitation, Fexcitation, F630630/F/F580580 was measuredwas measured
Both demonstrate that Both demonstrate that the spectral shift is a the spectral shift is a linear function of the linear function of the cell membrane cell membrane potentialpotential
Experimental Data
Raw Optical Signals
15
20
25
30
35
40
0 1 2 3 4 5
Time (s)
Am
plit
ude
(mV
) Long Short
Non-Ratioed Optical Signal
-100 -50 0 50 100 150 200
Time (ms)
Ratioed Optical Signal
-100 -50 0 50 100 150 200
Time (ms)
Intracellular Potential
-100 -50 0 50 100 150 200
Time (ms)
Comparisons between Systems
Conventional systemConventional system ~ 2m x 1m~ 2m x 1m Water Water cooledcooled gas laser (5W)gas laser (5W) Free space opticsFree space optics
Modular fibre systemModular fibre system ~~ 20cm x 10cm20cm x 10cm Solid state laser (50mW)Solid state laser (50mW) Modular fibre constructionModular fibre construction
Future Developments
High speed High speed spectrophotometerspectrophotometer
Possible removal of Possible removal of excitation light with excitation light with softwaresoftware
Study in the presence of Study in the presence of haemoglobinhaemoglobin
Other functional Other functional fluorescence imagingfluorescence imaging
Multifunctional studyMultifunctional study
Fluorescence Spectrum of di-4-ANEPPS under532nm Excitation
0
200
400
600
800
500 550 600 650 700 750 800
Wavelength (nm)(a
u)
Acknowledgement
Marsden FundMarsden Fund
FoRSTFoRST
University of AucklandUniversity of Auckland
RSNZ R H T Bates ScholarshipRSNZ R H T Bates Scholarship