Lecture 17

Tissue fluorescence (Part II)

Dimension reduction: Principal Component Analysis

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Component loadingsspectra337 nm

380 nm

460 nm

Spectroscopic analysis using PCA

• Uses full spectrum information to optimize sensitivity and specificity

• Relatively easy to implement (automated software)

• Provides no intuition with regards to the origin of spectral differences

Spectroscopic imaging: fluorescence ratio methods for

detection of lung neoplasia

B. Palcic et al, Chest 99:742-3, 1991

Detection of lung carcinoma in situ using the LIFE imaging

system

Courtesy of Xillix Technologies (www.xillix.com)

White light bronchoscopy Autofluorescence ratioimage

Fluorescence imaging based on ratio methods

• Wide field of view (probably a huge advantage for most clinical settings)

• Eliminates effects of distance and angle of illumination

• Easy to implement• Provides no intuition with regards to

origins of spectral differences

What are the origins of the observed differences?

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337 nm excitation358 nm excitation381 nm excitation

397 nm excitation412 nm excitation425 nm excitation

Collagen NADH

Collagen and NADH spectra are sufficiently distinct only for some excitation

wavelengths

337 nm excitation 358 nm excitation

Tissue absorption and scattering may affect significantly tissue

fluorescence• scattering

– elastic scattering• multiple scattering

• absorption– Hemoglobin, beta carotene

• fluorescence

• single scattering

epithelium

Connective tissue

Is hemoglobin absorption a problem?

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To get answer use

Monte Carlo simulations

Analytical Modeling

Monte Carlo simulations• Treat light as individual photons• Assign to each photon a probability of being

absorbed or being scattered in a particular direction

• Collect photons that make it out of the tissue• Relatively easy to model different light

delivery/collection geometries and multiple tissue layers

• Require a lot of time for acquiring statistically valid results

Analytical models

• Use an expression to describe the collected fluorescence as a function of tissue and light parameters

• Fast and easy to implement

• Require approximations and a priori information

fluorescence path

Tissue

reflectance path

Photon Migration-based Model

Müller et al. Applied Optics 40: 4633-4646, 2001

R() nwn

n1

Fxm n1

niwni

i0

n 1

EscapeProbability after n scattering events

Photon weight

n *e n Based on Monte Carlosimulations

as

snn aaw

,

Escape probability after i scattering events at x, followed by fluorescence at the (i+1)th event, followed by n-i-1 scatter events at m

We can recover the intrinsic fluorescence by combining fluorescence and reflectance measurements

fluorescence path

Tissue

reflectance path

Photon Migration-based Model

f = intrinsic fluorescenceF = observed fluorescenceR = reflectance = probe and anisotropy dependent parameters

Müller et al. Applied Optics 40: 4633-4646, 2001

inte

nsi

ty

wavelength (nm) wavelength (nm)

Model recovers intrinsic fluroscencelineshape and intensity in samples with

known optical properties!

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measured fluorescence intrinsic fluorescence

fluorophore

fluorophore + beads

Increasing amountof hemoglobin

Fast Excitation Emission Matrix (FastEEM) instrument

Optical MultichannelAnalyzer

Xe Flash L

amp

Tissue

Spectrograph

N 2 L

aser

Rapidly spinningdye cell wheel

Fiber Probecross-sectionalview

Collects fluorescence emission spectra at 11 laser excitation wavelengths between 337 and 610 nm and a white light reflectance spectrum in about 1s

In vivo tissue fluorectification...

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measured fluorescence

modeled intrinsic fluorescence

wavelength (nm)

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wavelength (nm)

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measured fluorescence Intrinsic fluorescence

Data from 4 esophageal varices: Intrinsic fluorescence is nicely recovered

patient 1/varix1patient 1/varix 2patient 1/varix 3patient 2/varix 1

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Intrinsic tissue fluorescence changes during dysplastic progression

wavelength (nm)

measured fluorescence

337 nm excitation

Non-dysplastic Barrett’s esophagus Low-grade dysplasiaHigh-grade dysplasia

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intrinsic fluorescence

Georgakoudi et al. Gastroenterology, 120: 1620-1629, 2001

Intrinsic tissue fluorescence changes during disease progression

Barrett’s esophagus Uterine cervix

Oral cavity artery

Normal

Diseased

337 nm excitation

Georgakoudi et al. Biomedical Photonics Handbook, CRC Press, ed. Vo Dinh, ch.31, 2003

EEM(HSIL) = * EEM(collagen) + *EEM(NAD(P)H)

Georgakoudi et al. Cancer Research 62: 682-687, 2002

=

b

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m)

Quantitative biochemical information is extracted from intrinsic tissue fluorescence

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collagen/(collagen+elastin)

Barrett’s esophagus cervix

Oral cavityCoronary artery

Normal

Diseased

Georgakoudi et al. Biomedical Photonics Handbook, CRC Press, ed. Vo Dinh, ch31, 2003

Model based fluorescence spectral analysis

• Utilizes full spectrum information• Model development may require

assumptions/approximations• Results are quantitative and provide

insights into the origins of spectral difference

• Instrument optimization• Disease understanding

Fluorescence as a PDT dose metric

1O2

3O2

C ollisionalQuenching

Ground StateTriplet Oxygen

Excited stateSinglet oxygen

Type II: Oxygen radicals

Type I: Free radicals

C YTOTOXIC ITY

Ground State So

Exited SingletState S1

Excited Triplet State T1A

bsorption

Fluorescence

Phosphorescence

1O2

3O2

C ollisionalQuenching

Ground StateTriplet Oxygen

Excited stateSinglet oxygen

Type II: Oxygen radicals

Type I: Free radicals

C YTOTOXIC ITY

Ground State So

Exited SingletState S1

Excited Triplet State T1A

bsorption

Fluorescence

Phosphorescence

Sensitizer fluorescence provides information on:•Sensitizer concentration •Production of cytotoxic moieties

Tailor PDT dosimetry to individual patients

Courtesy of J. Benavides, Wellman Center for Photomedicine

AURORA Dosimeter

The fiber optic bundle inserted in catheter with endoscope.Excitation wavelength: 405nmTuned to detect PPIX 635 nm fluorescence peak

Nitrogen Diode Laser

Fiber OpticBundle probe

PMT

405nmLongpass

filterLensNitrogen

Diode Laser

Fiber OpticBundle probe

PMT

405nmLongpass

filterLens

Fiber Bundle Tip: - One excitation/six collection 100 m core diameter fibers - Excitation-collection = 120 m - Bundle tip Diameter = 0.1’’

Courtesy of T. Hasan laboratory, Wellman Center for Photomedicine

Clinical Protocol

zz zz

Buccal mucosa fluorescencemeasurements

11 22 33

44 55 66

77

Patients receive a solution of 30 mg/kg body weight of ALA powder

Wait 4 hours

Patients received topical anesthesia of pharynx and intravenous conscious sedation

Pre-irradiation measurements and biopsies taken from Barrett’s and normal sites.

Balloon is inserted and continuous or fractionated light irradiation is administered

Post-irradiation measurements on previously tested spots

Continuous Light Dose Fractionated Light DoseFluence Rate: 150 mW/cm2 without fractionation (16 min and 40 seconds).Fluence: 150 J/cm2

Fluence Rate: 150 mW/cm2 with 60 second light/dark fractionation intervals.Fluence: 150 J/cm2

Courtesy of J. Benavides, T. Hasan, Wellman Center for Photomedicine

Fractionated Illumination

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Oral Cavity Squamous Barrett's

Flu

ores

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Results: PPIX PhotobleachingFRACTIONATED ( 3 PATIENTS)

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luor

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•Significant photobleaching in treatment area

•Higher photobleaching observed for continuous than for fractionated irradiation protocol

CONTINUOUS ( 4 PATIENTS)Pre-ALAPost-ALA, Pre PDTPost PDT

n=9

n=8 n=2

n=5 n=6n=5

n=6

n=9

n=4

n=6

n=5

n=5

n=8

n=6

n = number of sites

Courtesy of T. Hasan laboratory, Wellman Center for Photomedicine

Fluorescence photobleaching as a PDT dosimetry monitor

Courtesy of T.H. Foster, University of Rochester

404 nm: porphyrin fluorescence excitationHalogen lamp: reflectance spectrumLaser: PDT treatment

Correction of the Measured Fluorescence Using Reflectance at the Excitation and Emission Wavelengths:Model development/validation using tissue phantoms

fxm Fxm

Rx0.8Rm

as

salbedo

Tissue phantom: liquid or solid material with scattering and absorption properties, which mimics those of tissue

Porphyrin Spectra in Vivo are Complex and Variable Following ALA and Irradiation

Pre PDT 100 mW 5 mW

Rat skin200 mg/kg ALA514 nm irradiation

Photoproduct I Photoproduct II

DuringPDT

100 mW

Each spectrum takenAt 3 J cm-2 intervals

Spectra startingAt 15 J cm-2: photoproduct at 675 nm

Spectra starting at 10 J cm-2 at 2 J cm-2 intervals: photoproduct at 622 nm

Spectroscopy is Consistent with Greater PDT Effect at Low Irradiance

m-THPC Fluorescence in vivo Exhibits Two Phases Separated by an Irradiance-Dependent Discontinuity

1 mg/kg mTHPC 72 h prior to irradiation at 650 nm

As PDT progresses, m-THPC peak emission intensity decreasesThe rate at which this decrease occurs depends on the fluence rate of irradiation

Photofrin Fluorescence Spectra Exhibit Irradiance Dependent Features Following PDT in vivo

Photofrin Photoproducts But Not Fluorescence Photobleaching Appear to Report Biological Response

Fluorescence spectroscopy in PDT

• Enhance accuracy of sensitizer dose

• Indirect reporter of deposited cytotoxic dose

• Tailor dosimetry to individual patient physiology/biochemistry

Fluorescence life-time methods• Provide an additional dimension of information

missing in time-integrated steady-state spectral measurements

• Sensitive to biochemical microenvironment, including local pH, oxygenation and binding

• Lifetimes unaffected by variations in excitation intensity, concentration or sources of optical loss

• Compatible with clinical measurements in vivo

Courtesy of M.-A. Mycek, U Michigan

Fluorescence lifetime measurements

Autofluorescence lifetimes used to distinguish adenomatous from non-

adenomatous polyps in vivo

M.-A. Mycek et al.GI Endoscopy 48:390-4, 1998

Conclusion

• Fluorescence spectra provide a rich source of information on tissue state, which may be used to improve current methods of disease detection and treatment

• Fluorescence-based instrumentation is relatively simple, compact and compatible with clinical measurements