Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan...

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Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Dean Tai Bryan Caldwell Bryan Caldwell Ian LeGrice Ian LeGrice John Harvey John Harvey Sally Rutherford Sally Rutherford Bruce Smaill Bruce Smaill

Transcript of Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan...

Page 1: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

Modular Fiber Optic System for Intramural Functional

Fluorescence MeasurementDean TaiDean Tai

Bryan CaldwellBryan CaldwellIan LeGriceIan LeGriceJohn HarveyJohn Harvey

Sally RutherfordSally RutherfordBruce SmaillBruce Smaill

Page 2: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

Outline

Functional fluorescence imaging systemFunctional fluorescence imaging systemOptrodeOptrodeModular fiber fluorescence imaging Modular fiber fluorescence imaging

systemsystemResultsResultsFuFutureture Development Development

Page 3: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

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

Page 4: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

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

Page 5: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

Optrode

Page 6: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

Modular All-Fibre Imaging System

Solid state laserSolid state laser Fibre couplerFibre coupler

1-to-8 fibre splitter1-to-8 fibre splitter

Page 7: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

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%

Page 8: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

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

Page 9: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

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

Page 10: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

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

Page 11: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

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)

Page 12: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

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

Page 13: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

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)

Page 14: Modular Fiber Optic System for Intramural Functional Fluorescence Measurement Dean Tai Bryan Caldwell Ian LeGrice John Harvey Sally Rutherford Bruce Smaill.

Acknowledgement

Marsden FundMarsden Fund

FoRSTFoRST

University of AucklandUniversity of Auckland

RSNZ R H T Bates ScholarshipRSNZ R H T Bates Scholarship