biomedical raman spectroscopy

Biomedical Raman Spectroscopy

Jason T. Motz

Harvard Medical School and The Wellman Center for PhotomedicineMassachusetts General Hospital

11 April 2006

A New Type of Secondary Radiation

C. V. Raman and K. S. Krishnan, Nature, 121(3048): 501-502, March 31, 1928

If we assume that the X-ray scattering of the 'unmodified' type observed by Prof. Compton corresponds to the normal or average state of the atoms and molecules, while the 'modified' scattering of altered wave-length corresponds to their fluctuations from that state, it would follow that we should expect also in the case of ordinary light two types of scattering, one determined by the normal optical properties of the atoms or molecules, and another representing the effect of their fluctuations from their normal state. It accordingly becomes necessary to test whether this is actually the case. The experiments we have made have confirmed this anticipation, and shown that in every case in which light is scattered by the molecules in dust-free liquids or gases, the diffuse radiation of the ordinary kind, having the same wave-length as the incident beam, is accompanied by a modified scattered radiation of degraded frequency.

The new type of light scattering discovered by us naturally requires very powerful illumination for its observation. In our experiments, a beam of sunlight was converged successively by a telescope objective of 18 cm. aperture and 230 cm. focal length, and by a second lens was placed the scattering material, which is either a liquid (carefully purified by repeated distillation in vacuo) or its dust-free vapour. To detect the presence of a modified scattered radiation, the method of complementary light-filters was used. A blue-violet filter, when coupled with a yellow-green filter and placed in the incident light, completely extinguished the track of the light through the liquid or vapour. The reappearance of the track when the yellow filter is transferred to a place between it and the observer's eye is proof of the existence of a modified scattered radiation. Spectroscopic confirmation is also available.

Some sixty different common liquids have been examined in this way, and every one of them showed the effect in greater or less degree. That the effect is a true scattering, and secondly by its polarisation, which is in many cases quire strong and comparable with the polarisation of the ordinary scattering. The investigation is naturally much more difficult in the case of gases and vapours, owing to the excessive feebleness of the effect. Nevertheless, when the vapour is of sufficient density, for example with ether or amylene, the modified scattering is readily demonstrable.

Professor Sir C.V. Raman

1888-1970

The Nobel Prize in Physics 1930

"for his work on the scattering of light and for the discovery of the effect named after him"

First photographed Raman spectra Bangalore, India

The Raman Effect: Inelastic Scatteringhi

h(i-R)

Virtual Level

Rayleigh Raman (inelastic)(elastic) Scattering Scattering

Inelastic Scattering

• Energy transferred from incident light to molecular vibrations

• Emitted light has decreased energy (i<R)

difference in energy

Some Vibrations in Benzene

BreathingChubby CheckerKekule

400 600 800 1000 1200 1400 1600 18000

5x 104

Raman Shift (cm-1)

Evolution of Raman Spectroscopy• 1928~1960

– Minor experimental advances

• 1960– Invention of laser expands scope experiments

• 1980s: rapid technological advances– Fourier Transform spectroscopy– Charge Coupled Device (CCD) detectors– Holographic and dielectric filters– Near-Infrared (NIR) lasers

• Late 1980s1990s– Biomedical investigations– Advanced dispersive spectrometers

• 2000 – In vivo application– Optical fiber probes– Non-linear spectroscopy

Outline

• The Raman Effect– Theory– Techniques & Applications

• Biomedical Raman Spectroscopy– History– Excitation wavelength selection– Advantages of Raman spectroscopy

• Instrumentation– Laboratory– Clinical: optical fiber probes

• Case Study: Atherosclerosis– Disease background– Impact of Raman spectroscopy

• Frontiers

Classical Raman Physics

• Interaction between electric field of incident photon and molecule– Electric field oscillating with incident frequency vi:

– Induces molecular electric dipole (p):

• Proportional to molecular polarizability, – ease with which the electron cloud around a molecule

can be distorted

– Polarization results in nuclear displacement

0 cos(2 )i iE E t

0 cos 2 Rq q t

Classical Raman Physics

• For small distortions, polarizability is linearly proportional to the displacement

• Resultant dipole:

0 0 cos 2 ip E E t

Rayleigh Scattering

Anti-Stokes RamanStokes Raman

1 cos 2 cos 22 i R i RE q t t

Auto- IR Rayleigh Stokes Anti-Stokes NIRFluorescence Absorption Scattering Raman Scattering Fluorescence

Virtual Levels

0 n’1

Photo-Molecular Interactions

-2000 -1000 0 1000 20000

Raman Shift (cm-1)

Anti-StokesStokes

RayleighScattering

Classical Raman Physics• Raman scattering occurs only when the molecule is ‘polarizable’

• Raman intensity 4

– Classical dipole radiation– Stokes shifted Raman is more intense than anti-Stokes by Boltzmann factor:

• Consistent with other scattering phenomena, often reported in terms of cross-section ( [cm2]), or probability of scattering:

: density of molecules– dz: pathlength

i RA kT

0I I dz

Characteristics of Raman Scattering

• Very weak effect– Only 1 in 107 photons is Raman scattered– NIR elastic scattering in tissue:– NIR absorption in tissue:– Red absorption in tissue or water:– Raman scattering in tissue or water:

• True scattering process – Virtual state is a short-lived distortion of the electron cloud

which creates molecular vibrations < 10-14 s

• Strong Raman scatterers have distributed electron clouds– C=C -bonds

'1/ 1s mm

1/ 10a cm 1/ 5a m

1/ 3R km

Quantum Mechanics: Normal Modes• Only certain vibrational frequencies and atomic

displacements allowed– Linear molecules: 3N-5– Non-linear molecules: 3N-6

• Examples– Stretching between 2 atoms– Symmetric and asymmetric stretching with 3 atoms– Bending amongst 3 atoms– Out of plane deformations

• Vibrational energies are sensitive to– Atomic mass – Molecular structure and geometry– Bond strength– Bond order– Environment– Hydrogen bonding

Units & Dimensional Analysis

• Spectroscopic frequencies reported in wavenumbers [cm-1], proportional to transition energy :

• Raman frequencies are independent of excitation wavelength and reported as shifts– Wavenumbers relative to excitation frequency:

1Ehc c

Units & Dimensional Analysis

• Example– NIR excitation at 830 nm: 12,048 cm-1 – Typical Raman shift: ~1000 cm-1

R = 905 nm

– Sharp biological Raman linewidths ~10 cm-1 FWHMR= 0.69 nm

Raman Spectrum of Cholesterol

Hanlon et al. “Prospects for in vivo Raman spectroscopy,” Phys Med Biol 45: R1 (2000)

Specific Raman Spectroscopic Techniques

• Non-resonant Raman spectroscopy– Visible– Near-infrared

• (UV) Resonance Raman spectroscopy • Raman microscopy/imaging• Fiber optic sampling• Time resolved (pulsed) Raman spectroscopy• High-wavenumber Raman spectroscopy• SERS: Surfaced Enhanced Raman Spectroscopy• Non-linear Raman spectroscopy

– CARS: Coherent Anti-Stokes Raman Spectroscopy

General Applications of Raman Spectroscopy

• Structural chemistry• Solid state• Analytical chemistry• Applied materials analysis• Process control• Microspectroscopy/imaging• Environmental monitoring• Biomedical

Outline

• Frontiers

History of Biological Raman Spectroscopy

• 1970: Lord and Yu record 1st protein spectrum from lysozyme using HeNe excitation

• Evolution to NIR excitation– Decreased fluorescence– Increased penetration (mm)

• 1980s: – FT Raman with Nd:YAG and cooled InGaAs detectors

• long collection times (30 min)– Clarke (1987-1988): visible excitation of arterial calcium hydroxyapatite

and carotenoids

• 1990s, advances in:– Lasers– Detectors– Dispersive spectrometers– Filters– Chemometrics

UV, Visible, and NIR Excitation

Raman signals have a constant shift can vary excitation wavelength• UV: resonance enhanced, R<F, photo damage, low penetration• Visible: Raman -4, fluorescence overlaps with Raman signal• NIR: low fluorescence, deep penetration, Raman -4

UV, Visible, and NIR Applications

• UVRR– Biological macromolecules: nucleic acids, proteins, lipids– Organelles, cells, micro-organisms, bacteria, phytoplankton

neurotoxins, viruses– Clinically limited: photomutagenicity

• Visible– Cells (minimal fluorescence)– DNA in chromosomes, pigment in granulocytes and

lymphocytes, RBCs, hepatocytes– First artery studies: hydroxyapatite and carotenoids

(Clarke 1987, 1988)• NIR

– Hirschfeld & Chase, 1986: FT-Raman– Tissue: artery, cervix, skin, breast, blood, GI, esophagus,

brain tumor, Alzheimer’s, prostate, bone

Raman Spectra: Fingerprinting a Molecule

• Raman spectra are molecule specific

• Spectra contain information about vibrational modes of the molecule

• Spectra have sharp features, allowing identification of the molecule by its spectrum

Examples of analytes found in blood which are quantifiable with Raman spectroscopy

• Narrow vibrational bands are chemical specific and rich in information

• Freedom to choose excitation wavelength– minimize unwanted tissue fluorescence– optimize sampling depth– utilize CCD technology

800 1000 1200 1400 1600 1800-0.4

Phenylalanine

Amide III

CH2 bend

Carotenoid

Ester linkageSterol ring

Phosphate stretch

Raman Shift (cm-1)

350 mW

Spectroscopic Advantages of NIR Raman

100 200 500 1000 2 000

m-1) H2O

HbOHbO

Visible

NIR ExcitationMelanin

Wavelength (nm)

Virtual Level

Fluorescence Raman (inelastic)Scattering

Diagnostic Advantages of Raman Spectroscopy

• Wavelength selection

• No biopsy required

• Directly measures molecules– Small concentrations– Chemical composition– Morphological analysis

• Quantitative analysis

• In vivo diagnosis

Outline

• Frontiers

Laser Sources for Raman Spectroscopy

Source Wavelength (nm)

Ar+ 488.0, 514.5

Kr+ 530.9, 647.1

He: Ne 632.8

Ti: Al2O3 (cw) 720-1000

Diode (InGaAs) 785, 830

Nd:YAG 1064

In Vitro Experimental Raman SystemCCD

Collimating lenses

s filte

Notch filter

Ti: sapphirelaser

Argon ion pump laser

NIR excitation (830 nm)

f/4Spectrograph

Dichroic beam-splitter

Confocalpinhole

Motorized translation stage

CCD Camera

In Vitro Turbid Liquid Analysis

ppm accuracy for precise quantitative measurements

Hanlon et al. “Prospects for in vivo Raman spectroscopy,” Phys Med Biol 45: R1 (2000)

Clinical Raman Systems

830 nm bandpassfilter

notch filter

holographic grating

shutterDiode Laser

Current Raman Instrumentation

• Laser diodes– Compact– Stable narrow line– NIR

• High throughput spectrographs (f/1.8)• Holographic elements

– Bandpass filters (eliminates spontaneous emission of lasing medium)– Notch filters (106 rejection of Rayleigh scattered laser line)– Large area, highly efficient transmission gratings

• CCD detectors– High QE (back-thinned, deep-depletion)– Low noise (LN2 cooled)– Multichannel detection

• High throughput, filtered fiber optics probes

• NIR FT and scanning PMT systems no longer useful

Early in vivo Data

• Simple 6-around-1 optical fiber probe• 100 mW excitation, 3 second collection

Problems

1. Fiber background• Distorts signal• Adds shot-noise

2. Low signal collection• Raman effect is weak• Tissue is highly diffusive

Optical Fiber Probes

Fiber background NA2

Solution #1: Reduce Fiber BackgroundFiber background produced equally in excitation and collection fibers

• Excitation laser of power Po generates Raman scattered light from tissue

• Posxlx: fraction of laser light Raman scattered and transmitted by excitation fiber*

• Bx=Posxlxes: Raman background detected from excitation fiber

– es: fraction of light elastically scattered (and collected) from sample • Poes: intensity of scattered excitation light gathered by collection fibers

• Bc=Poessclc: intensity of background generated and transmitted by collection fibers*

• BT=Bx+Bc=Poes(sxlx+sclc)

Tissue Sample

x c* NA2

From McCreery RL “Raman Spectroscopy for Chemical Analysis,” 2000.

Excitation laser

Tissue Raman

Fiber background

Tissue Sample

Filter Transmission

0 500 1000 15000

Raman Shift (cm-1)

Collection FilterExcitation Filter

Region of Interest

Problems

Solutions

1. Micro-optical filters• Short-pass excitation filter• Long-pass collection filter

2. Optimize optical design• Characterize distribution

of Raman light in tissue• Define optimal geometry• Design collection optics

Solution #1: Filtering

Excitation Light Diffusing Through Tissue

Monte Carlo

Experimental

Problems

Solutions

1. Micro-optical filters• Short-pass excitation filter• Long-pass collection filter

2. Optimize optical design• Characterize distribution

of Raman light in tissue• Define optimal geometry• Design collection optics

Solution #2: Optical Design

Raman Probe Design Goals

• Restricted geometry for clinical use– Total diameter ~2mm for access to coronary arteries– Flexible– Able to withstand sterilization

• Designed to work with 830 nm excitation

• High throughput– Data accumulation in 1 or 2 seconds– Safe power levels– SNR similar to open-air optics laboratory system– Accurate application of models

Raman Probe Design

retainingsleeveball lens

1.75 mm

collection fibers

long-passfilter tube

metal sleeve

aluminum jacket

excitation fiber

short-passfilter rod

Single Ring Probe has 15 FibersMotz et al. Appl Opt 43: 52 (2004)

0 500 1000 15000

Raman Shift (cm-1)

Single Ring, sapphire lensLab system

Calcified Aorta

Outline

• Frontiers

The Burden of Cardiovascular Disease†

• 71,300,000 people in United States afflicted

• 910,600 deaths per year– 1 out of every 2.7 deaths

• Coronary artery disease claims 653,000 lives annually– 1 out of every 5 deaths– Economic cost: greater than $142.5 billion

†American Heart Association, Heart and Stroke Statistics-2006 Update

Arterial Anatomy

Intima

Adventitia

TAtheroma

Fibrous Cap

Normal Mildly Atherosclerotic Plaque Ruptured Plaque

• Intima: innermost layer of arterial wall• composed of a single layer of endothelia cells in normal artery• region of artery involved in atherosclerotic disease

• Media: arterial layer composed primarily of smooth muscle cells• constricts and dilates to control blood flow• in large arteries (e.g. aorta) this layer is largely composed of elastin

• Adventitia: outermost layer of arterial wall• connective tissue and fat

T: thrombusNC: necrotic core

Some Current Challenges in Cardiology

• Evaluation and development of therapeutics

• Etiology of atherosclerosis

• Mechanisms of re-stenosis– Post-angioplasty– Transplant vasculopathy

• Detection of vulnerable atherosclerotic plaques– Prediction/prevention of cardiac events

Vulnerable Plaques

• Account for majority of sudden cardiac death• Frequently occur in clinically silent vessels

– <50% stenosis• Effective treatments unknown • Characterized by:

– Biochemical changes– Foam cells– Lipid pool – Inflammatory cells– Thin fibrous cap (<65 m)

• Currently undetectable

Standard Diagnostic Techniques

• Angiography– Severity of stenosis, thrombosis, dense calcifications– Provides no biochemical information

• Angioscopy– Surface features of plaque, including color– No information of sub-surface features

• Histopathology– Biochemical and morphological information– Requires excision of tissue

Emerging Diagnostic Techniques

• Magnetic resonance imaging

• External ultrasound

• Positron emission tomography

• Electron beam computed tomography

• Thermography

• Elastography

• Intravascular ultrasound

• Optical coherence tomography

Non-Invasive

Spectroscopic Diagnostic Techniques

• NIR Absorption spectroscopy– Inhibited by water absorption– Broad spectral features

• Fluorescence spectroscopy– Limited chemical information– Broad spectral features

• Raman Spectroscopy– Quantitative biochemical information– Morphological analysis

In Vitro Experimental Methods

• Macroscopic Raman Spectroscopy– 1 mm3 volumes of excised tissue are examined

• 100-350 mW excitation with 830 nm laser light• 10 - 100 s collection times

– Comparison with histopathology for disease classification– Principal Component Analysis

53 Image from medstat.med.utah.edu/WebPath/webpath.html

Raman Spectral Pathology of Atherosclerosis

normal artery

lipid-rich plaque

calcified plaque

• collagen• elastin• actin

• cholesterol• -carotene• proteins

• Ca hydroxyapatite• proteins

Raman Spectral Modeling

• Goal: Diagnose disease by analyzing the complex macroscopic spectra (R) obtained from biopsy samples

• Strategy: Develop a library of microscopic or chemical basis spectra (B) that compose the macroscopic data

• Implementation: Use ordinary least squares fitting to determine a weighted linear combination of basis spectra to

evaluate the biopsies

R()artery = wcollagenB()collagen+ wcholesterolB()cholesterol+ wcalcificationB()calcification+…

Potential Features for Spectral Identification

CollagenElastinActin

Adventitial fat-caroteneFoam cellsCholesterol

Necrotic coreCalcificationHemoglobin

Fibrin

In Vitro Experimental Methods

• Macroscopic Raman Spectroscopy– 1 mm3 volumes of excised tissue are examined

• 100-350 mW excitation with 830 nm laser light• 10 - 100 s collection times

– Comparison with histopathology for disease classification– Principal Component Analysis

• Confocal Microscopic Raman Spectroscopy– ~(2x2x2) m3 sampling volume of microscopic structures

• 100 mW excitation of 6 m thick sections with 830 nm laser light• 10-360 s collection times

– Development of morphological model– Spectroscopic mapping of tissue sections

Atherosclerosis In Vitro: Confocal Microscopy

AdventitiaMediaIntima ~(2x2x2) m3 Sampling Volume

800 1000 1200 1400 1600 1800

Raman Shift (cm-1)

Elastic Lamina

800 1000 1200 1400 1600 1800-0.4

ity (a

Raman Shift (cm-1)

Foam Cell

800 1000 1200 1400 1600 1800

Raman Shift (cm-1)

Smooth Muscle Cell0.1 mm

Coronary Artery Morphological Structures

Buschman HPJ, et al. Cardiovascular Pathology 10(2), 69-82 (2001)

800 1200 1600

Raman shift (cm-1)

800 1200 1600

sity (

Raman shift (cm-1)

Calcified Plaque

Residual

Macroscopic Data

MicroscopicModel Fit

Buschman HPJ, Motz JT, et al. Cardiovascular Pathology 10(2), 59-68 (2001)

800 1200 1600

Raman shift (cm-1)

Normal Coronary Artery Non-Calcified PlaqueMorphological Model of Coronary Arteries

61800 1000 1200 1400 1600 1800

-1.2-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.01.2

Raman Shift (cm-1)

800 1000 1200 1400 1600 1800-1.2-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.01.2

Raman Shift (cm-1)

Morphological Assay of Coronary Arteries

Mildly Calcified Plaque Structure ContributionCollagen 39%Cholesterol 10%Calcification 11%Elastic Lamina 0%Fat 28%Foam Cell / Core 0%-Carotene 0%Smooth Muscle 12%

Structure ContributionCollagen 20%Cholesterol 6%Calcification 0%Elastic Lamina 6%Fat 38%Foam Cell / Core 0%-Carotene 3%Smooth Muscle 27%

Normal Coronary Artery

Diagnostic Database

• 165 intact samples from explanted hearts– Biopsies snap frozen until examination– 2 data sets

• Calibration (n=97)• Prospective (n=68)

• Three tissue categories determined by histopathology– Non-atherosclerotic– Non-calcified plaque– Calcified plaque

Coronary Artery Disease Classification:A Prospective Study

Buschman HPJ, Motz JT, et al. Cardiovascular Pathology 10(2), 59-68 (2001)

Raman Morphometry of Coronary Artery

Buschman HPJ, et al. Cardiovascular Pathology 10(2), 59-68 (2001)

Clinical Data: Methods

• Peripheral vascular surgery– Femoral bypass– Carotid endarterectomy

• Laser power calibration set with Teflon– ~100 mW (82-132mW)

• OR room lights turned off as during angioscopy

• Spectra collected for a total of 5 seconds– 20 accumulations of 0.25s each– Probe held normal to arterial wall

• Analysis of 1s and 5s data – Additional model components: sapphire, epoxy, water, HbO2

All data presented are integrated for only 1 second

Clinical Data: Intimal Fibroplasia (Normal)

0.4 mm

800 1000 1200 1400 1600 1800

Raman Shift (cm-1)

DataFitResidual

Motz JT et al., J Biomed Opt 11(2): 021003

Clinical Data: Atheromatous Plaque

0.4 mm

0.1 mm

800 1000 1200 1400 1600 1800

Raman Shift (cm-1)

DataFitResidual

1.8 mm deepcalcification

Clinical Data: Calcified Plaque

800 1000 1200 1400 1600 1800

Raman Shift (cm-1)

DataFitResidual

Clinical Data: Ruptured Plaque

0.4 mm

0.1 mm

800 1000 1200 1400 1600 1800

Raman Shift (cm-1)

DataFitResidual

Clinical Data: Thrombotic Plaque

0.4 mm

0.1 mm

800 1000 1200 1400 1600 1800

Raman Shift (cm-1)

DataFitResidual

Clinical Data: Representative Analysis

Model Component IntimalFibroplasia

Atheromatous Plaque

Calcified Plaque

Ruptured Plaque

Thrombotic Plaque

Collagen (%) 9 0 7 0 0

Cholesterol (%) 0 44 2 27 14

Calcification (%) 0 16 71 1 12

Elastic Lamina (%) 0 4 3 0 0

Adventitial Fat (%) 50 13 0 1 0

Lipid Core (%) 13 16 0 0 0

-Carotene (%) 0 7 4 23 13

Smooth Muscle (%) 28 0 12 47 61

Hemoglobin (a.u.) 3 0 0 13 27

Raman Spectroscopy in Cardiology

• Clinical contributions– Detection of vulnerable plaque (Prediction and Prevention)

• Collagen content fibrous cap thickness• Chemical composition of plaques• Identification of mechanical instabilities

– Evaluation and selection of interventional methods• Drug therapy• Restenosis of bypassed vessels

– Guidance for laser ablation therapy• Basic science

– Etiology: monitoring of chemical and morphological changes during disease progression

– Differences in diabetic atherosclerosis

Application To Other Diseases

100 mW excitation, 1 second collection

800 1000 1200 1400 1600 1800

Raman Shift (cm-1)

Data Fit Residual

Normal Breast Tissue

800 1000 1200 1400 1600 1800-1.5

Raman Shift (cm-1)In

ity (a

Data Fit Residual

Malignant Breast Tumor

Outline

• Frontiers

Frontier Investigations: High-Wavenumber Raman

www.sigma.com

Advantages• Higher Raman signal• Lower fluorescence• No fiber background• Distinguishes cholesterol esters

Disadvantages• Broader lineshapes• Smaller spectral region• Mostly limited to lipids• No calcification signalc

Cholesterol

High-Wavenumber Raman Spectroscopy

Koljenovic S et al., J Biomed Opt 10(3): 031116 (2005)

Cholesteryl palmitate

Cholesteryl linoleate

Triolein

Collagen

Glycogen

Normal Bladder

High-Wavenumber H&Elp: lamina propriau: urothelium

Frontier Investigations: Pulsed Excitation

0 5 10 15 20 25 30 35 400

Time (ns)

Remitted Intensity with Pulsed Excitation

Laser PulseRamanRayleighFluorescence

• 80 MHz repetition rate• 2 ns fluorescence lifetime• Rayleigh scattering ~t-3/2

• Raman scattering ~t-1/2

0 10 20 30 40 50 600

Time (ps)

Remitted Intensity with Pulsed Excitation

Laser PulseRamanRayleighFluorescence

Decay kinetics based on work of Everall N et al., Appl Spectrosc 55(12): 1701 (2001)

Frontier Investigations: Pulsed Excitation

Martyshkin DV et al., Rev Sci Instr 75(3): 630 (2004)

Conclusions

• Raman spectroscopy ‘fingerprints’ molecules by characterizing interactions between photons and molecular vibrations

• Near-infrared excitation is preferred for biomedical applications

• Recent optical fiber probe developments allow accurate real-time analysis in vivo

• New areas of research are promising for widespread clinical application

References

• McCreery RL. Raman Spectroscopy for Chemical Analysis. Wiley-Interscience, New York, 2000.

• Ferraro JR, Nakamoto K, and Brown CW. Introductory Raman Spectroscopy 2nd ed. Academic Press, Boston, 2003.

• Hanlon EB, et al. “Prospects for in vivo Raman spectroscopy,” Phys Med Biol 45: R1-R59 (2000).

• Mahadevan-Jensen A and Richards-Kortum R. “Raman spectroscopy for the detection of cancers and precancers,” J Biomed Opt 1:31-70 (1996).

• Utzinger U and Richards-Kortum R. “Fiber optic probes for biomedical spectroscopy,” J Biomed Opt 8: 121-147 (2003).

biomedical raman spectroscopy

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