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Biomedical Raman Spectroscopy

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					Biomedical Raman Spectroscopy
Jason T. Motz
Harvard Medical School and The Wellman Center for Photomedicine Massachusetts 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. 2

The Nobel Prize in Physics 1930

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

Professor Sir C.V. Raman

1888-1970

3

First photographed Raman spectra

Bangalore, India

The Raman Effect: Inelastic Scattering
hi h(i-R)
3 2 1 0

hi

S1

Inelastic Scattering

Virtual Level

Energy

• Energy transferred from incident light to molecular vibrations • Emitted light has decreased energy (i<R)

3

2
1 0

Rayleigh (elastic) Scattering
4

Raman (inelastic) Scattering

S0

difference in energy

Some Vibrations in Benzene
Kekule Chubby Checker Breathing

Intensity (CCD Counts)

5 4 3 2 1

x 10

4

0 400

600

800

5

1000 1200 1400 -1 Raman Shift (cm )

1600

1800

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 1980s1990s
– Biomedical investigations – Advanced dispersive spectrometers

• 2000 
– In vivo application – Optical fiber probes – Non-linear spectroscopy
6

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
7

Classical Raman Physics • Interaction between electric field of incident photon and molecule
– Electric field oscillating with incident frequency vi:
E i  E 0 cos(2  i t )

– Induces molecular electric dipole (p):
p E

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

– Polarization results in nuclear displacement
q  q 0 cos  2 R t 
8

Classical Raman Physics • For small distortions, polarizability is linearly proportional to the displacement
     0    q 0  ...  q 0

• Resultant dipole:
p   E   0 E 0 cos  2  i t  
1

Rayleigh Scattering

   E 0 q0   cos  2   i   R  t   cos  2   i   R  t      2 q 0 





Anti-Stokes Raman
9

Stokes Raman

Photo-Molecular Interactions
100
Scattering Rayleigh

3
Intensity

80 60 40 20 Anti-Stokes 0 -2000
Stokes

2 1 0

n1

-1000 0 1000 Raman Shift (cm-1)

2000

2 1 0 n‟1

Energy

Virtual Levels

E=hR
2 1 0

AutoFluorescence
10

IR Absorption

Rayleigh Scattering

Stokes Anti-Stokes Raman Scattering

NIR Fluorescence

n0

Classical Raman Physics
• Raman scattering occurs only when the molecule is „polarizable‟
 0

• Raman intensity  4

dq

– Classical dipole radiation – Stokes shifted Raman is more intense than anti-Stokes by Boltzmann factor:
IA IS  i  R     e  i  R 
4 h R kT

• Consistent with other scattering phenomena, often reported in terms of cross-section ( [cm2]), or probability of scattering:
I  I 0  dz

– : density of molecules – dz: pathlength
11

Characteristics of Raman Scattering
• Very weak effect
– – – – – Only 1 in 107 photons is Raman scattered NIR elastic scattering in tissue: 1 /  s'  1m m NIR absorption in tissue: 1 /  a  10 cm Red absorption in tissue or water: 1 /  a  5 m Raman scattering in tissue or water: 1 /  R  3 km

• 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
12

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
– – – – – –
13

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 :
 
E hc 


c

E  h c  



1



• Raman frequencies are independent of excitation wavelength and reported as shifts
– Wavenumbers relative to excitation frequency:
R 
14

1

i



1

R

Units & Dimensional Analysis • Example
– NIR excitation at 830 nm: 12,048 cm-1 – Typical Raman shift:  R ~1000 cm-1
• R = 905 nm

– Sharp biological Raman linewidths ~10 cm-1 FWHM
• R= 0.69 nm

15

Raman Spectrum of Cholesterol

16

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

• • • • • • •
17

(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 • • • • • • • •
18

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

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
21

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:
– – – – –
22

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
23

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
24

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

Spectroscopic Advantages of NIR Raman • Narrow vibrational bands are chemical specific and rich in information • Freedom to choose excitatione nwavelength CH b d
1 .0

Molar extinction coefficient  (10-3M-1cm-1); H20 (cm-1)

In te n s ity (a .u .)

– minimize punwanted tissue fluorescence 0 .8 P h o s h a te s tre tc h 3 C a ro te n o id 2 – optimize sampling depth 0 .6 1 106 A m id e III C=C – utilize CCD technology Visible 0 .4 H O 0 2
Energy
104 102 1
n1
0 .2 0 .0 -0 .2 -0 .4 800 1 2000 0 0 1200 1400

2

Melanin

NIR Excitation Virtual Level

350 mW 1 min

S te ro l rin g

HbO P h e n yla la n in e

3

E s te r lin k a g e 2
1 1800

10-2 100
26

HbO

1600 500 1000 0 -1 n0 FluorescenceS h ift Raman )(inelastic) R a m a n Wavelength (nm) (c m

2 000

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
27

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
28

Laser Sources for Raman Spectroscopy
Source Ar+ Kr+ He: Ne Wavelength (nm) 488.0, 514.5 530.9, 647.1 632.8

Ti: Al2O3 (cw)
Diode (InGaAs) Nd:YAG
29

720-1000
785, 830 1064

In Vitro Experimental Raman System
Argon ion pump laser Dichroic beam-splitter Ti: sapphire laser NIR excitation (830 nm)

Notch filter Band-pass filter Collimating lenses

CCD

f/4 Spectrograph Confocal pinhole
CCD Camera

30

Motorized translation stage

In Vitro Turbid Liquid Analysis

ppm accuracy for precise quantitative measurements
31

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

Clinical Raman Systems
830 nm
shutter

Diode Laser

bandpass filter

holographic grating

notch filter CCD
32

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
33

Early in vivo Data
• Simple 6-around-1 optical fiber probe • 100 mW excitation, 3 second collection
1500 500 -500 -5000 -1500 -2500 -3500 2000 -10000

a

5000

b

0

*

1600

1200

800

-15000 2000

1600

1200

800

Raman shift
34

Raman shift

Optical Fiber Probes
Problems
Fiber background  NA2

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

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

35

Solution #1: Reduce Fiber Background
Fiber 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) Excitation laser
*

NA2

Tissue Raman Fiber background

x

c

x

c

From McCreery RL “Raman Spectroscopy for Chemical Analysis,” 2000.
36

Tissue Sample Sample Tissue

Filter Transmission
100

80
Transmission (%)

Region of Interest
60 Collection Filter Excitation Filter

40

20

0 0
37

500 1000 -1 Raman Shift (cm )

1500

Solution #1: Filtering
Problems Solutions 1. Micro-optical filters
• • Short-pass excitation filter Long-pass collection filter

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

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

2. Optimize optical design
• • • Characterize distribution of Raman light in tissue Define optimal geometry Design collection optics

38

Excitation Light Diffusing Through Tissue
Monte Carlo Experimental

39

1 mm

Solution #2: Optical Design
Problems 1. Fiber background
• • Distorts signal Adds shot-noise

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

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

2. Optimize optical design
• • • Characterize distribution of Raman light in tissue Define optimal geometry Design collection optics

40

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
– – – –
41

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
collection fibers aluminum jacket excitation fiber long-pass filter tube 1 mm metal sleeve short-pass filter rod retaining sleeve
42

0.55 0.70 1.75 mm

2 mm

ball lens

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

Calcified Aorta
800 700 Single Ring, sapphire lens Lab system

Intensity (CCD counts/mW/s)

600 500 400 300 200 100 0 0 500 1000 Raman Shift (cm-1) 1500

43

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
44

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

45

†

American Heart Association, Heart and Stroke Statistics-2006 Update

Arterial Anatomy
Normal Mildly Atherosclerotic Plaque Ruptured Plaque

Media Fibrous Cap Atheroma Intima

Lumen

T NC

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

T: thrombus NC: necrotic core

• 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 46
• connective tissue and fat

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
47

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
48

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
49

Emerging Diagnostic Techniques
• Magnetic resonance imaging
• External ultrasound • Positron emission tomography • Electron beam computed tomography • Thermography
Non-Invasive

• Elastography
• Intravascular ultrasound

• Optical coherence tomography
50

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
51

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

52

Raman Spectral Pathology of Atherosclerosis
1 .0 0 .8 0 .6 0 .4 0 .2 0 .0 -0 .2
1 .2 1 .0

• Ca hydroxyapatite • proteins

800

1000 1200 1400 1600 1800

In te n s ity (a .u .)

0 .8 0 .6 0 .4 0 .2 0 .0 -0 .2 -0 .4 1 .2

• cholesterol • -carotene • proteins

800

1000 1200 1400 1600 1800

1 .0 0 .8 0 .6 0 .4 0 .2 0 .0 -0 .2 -0 .4 -0 .6

• collagen • elastin • actin

800

1000 1200 1400 1600 1800
-1

53

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

R a m a n S h ift (c m )

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+…
54

Potential Features for Spectral Identification
Collagen Elastin Actin Adventitial fat -carotene Foam cells Cholesterol Necrotic core Calcification Hemoglobin Fibrin

56

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 – Development of morphological model – Spectroscopic mapping of tissue sections

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

57

Atherosclerosis In Vitro: Confocal Microscopy
0 .8 0 .6

Foam Cell

0 .8 0 .6 0 .4

Elastic Lamina

In te n s ity (a .u .)

In te n s ity (a .u .)
800 1000 1200 1400
-1

0 .4 0 .2 0 .0 -0 .2 -0 .4

0 .2 0 .0 -0 .2 -0 .4

0.1 mm

1600

1800

800

1000

1200

1400
-1

1600

1800

R a m a n S h ift (c m )

R a m a n S h ift (c m ) 0 .8 0 .6 0 .4

Smooth Muscle Cell

In te n s ity (a .u .)

0 .2 0 .0 -0 .2 -0 .4 800 1000 1200 1400
-1

1600

1800

58

Intima

Media

Adventitia

R a m a n S h ift (c m )

~(2x2x2) m3 Sampling Volume

Coronary Artery Morphological Structures
Smooth Muscle Cell
-Carotene Crystal

Foam Cell/Core

Intensity (a.u.)

Adventitial Fat Elastic Lamina Calcification Cholesterol Crystal Collagen

800

1000

1200

1400
-1

1600

1800

Raman Shift (cm )
59

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

Morphological Model of Coronary Arteries
Normal Coronary Artery Non-Calcified Plaque

Intensity (a.u.)

Calcified Plaque
8 00 12 00 16 00
800 1200 1600

R an shift (cm-1) am

Intensity (a.u.)

Raman shift (cm-1)

Intensity (a.u.)

Macroscopic Data Microscopic Model Fit
800 1 200
-1 Ram shift (cm ) an

1 600

Residual

60

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

Morphological Assay of Coronary Arteries
Normal Coronary Artery
1 .2 1 .0 0 .8 0 .6 0 .4 0 .2 0 .0 -0 .2 -0 .4 -0 .6 -0 .8 -1 .0 -1 .2 800

1000

1200

1400
-1

1600

1800

Structure Collagen Cholesterol Calcification Elastic Lamina Fat Foam Cell / Core -Carotene Smooth Muscle Structure Collagen Cholesterol Calcification Elastic Lamina Fat Foam Cell / Core -Carotene Smooth Muscle

Contribution 20% 6% 0% 6% 38% 0% 3% 27% Contribution 39% 10% 11% 0% 28% 0% 0% 12%

In te n s ity (a .u .)

R a m a n S h ift (c m )

Mildly Calcified Plaque
1 .2 1 .0 0 .8 0 .6 0 .4 0 .2 0 .0 -0 .2 -0 .4 -0 .6 -0 .8 -1 .0 -1 .2 800 1000 1200 1400
-1

In te n s ity (a .u .)

1600

1800

61

R a m a n S h ift (c m )

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

62

Coronary Artery Disease Classification:
A Prospective Study
1.0
Normal Artery Non-Calcified Plaque Calcified Plaque Punctate Calcification

0.8

Calcification

0.6

0.4

3  error zone

0.2

0.0 0.0

0.1

0.2

0.3

0.4

0.5

0.6

CholesterolNCR + Lipid CoreNCR
63

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

Raman Morphometry of Coronary Artery
Relative Fit Coefficient (Mean +/- SEM)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
Normal Coronary Artery Non-Calcified Plaque Calcified Plaque

64

ch ol es te ro l ca lci f ic at io n el as tic la m in a ad fo ve am nt ita ce lf ll / at ne cr ot ic sm co oo re th m us be cle ta -c ce ar ll ot en e cr ys ta l

co

lla ge n

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
65

Clinical Data: Intimal Fibroplasia (Normal)
0.4 mm
1 Data Fit Residual 0.5

Intensity (a.u.)

0

-0.5

-1 800 1000 1200 1400 Raman Shift (cm-1) 1600 1800

66

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

Clinical Data: Atheromatous Plaque

1 Data Fit Residual 0.5

1.8 mm deep calcification

Intensity (a.u.)

0

0.4 mm
-0.5

0.1 mm
800 1000 1200 1400 Raman Shift (cm-1) 1600 1800

67

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

Clinical Data: Calcified Plaque
50 m
1 Data Fit Residual 0.5

Intensity (a.u.)

0

-0.5

800

1000

1200 1400 Raman Shift (cm-1)

1600

1800

68

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

Clinical Data: Ruptured Plaque
1 Data Fit Residual 0.5

Intensity (a.u.)

0

-0.5

-1

-1.5 800 1000 1200 1400 Raman Shift (cm-1) 1600 1800

0.1 mm

0.4 mm

69

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

Clinical Data: Thrombotic Plaque
0.4 mm
1 Data Fit Residual

0.5

Intensity (a.u.)

0

-0.5

-1

0.1 mm

-1.5 800 1000 1200 1400 Raman Shift (cm-1) 1600 1800

70

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

Clinical Data: Representative Analysis
Model Component Intimal Atheromatous Calcified Ruptured Thrombotic Plaque Plaque Plaque Plaque Fibroplasia 9 0 0 0 44 16 7 2 71 0 27 1 0 14 12

Collagen (%) Cholesterol (%) Calcification (%)

Elastic Lamina (%)
Adventitial Fat (%) Lipid Core (%) -Carotene (%) Smooth Muscle (%) Hemoglobin (a.u.)
71

0
50 13 0 28 3

4
13 16 7 0 0

3
0 0 4 12 0

0
1 0 23 47 13

0
0 0 13 61 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
72

Application To Other Diseases
Normal Breast Tissue
Data Fit Residual 0.5

1

1

Malignant Breast Tumor
Data Fit Residual

0.5

Intesnity (a.u.)

Intesnity (a.u.)
800 1000 1200 1400 Raman Shift (cm-1) 1600 1800

0

0

-0.5

-1 -0.5 -1.5 800 1000 1200 1400 Raman Shift (cm-1) 1600 1800

100 mW excitation, 1 second collection
73

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
74

Frontier Investigations: High-Wavenumber Raman
Cholesterol

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

75

www.sigma.com

High-Wavenumber Raman Spectroscopy

Normal Bladder DNA

Glycogen
Collagen Actin

High-Wavenumber Cholesteryl palmitate Cholesteryl linoleate Triolein
76

H&E
lp: lamina propria u: urothelium

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

Frontier Investigations: Pulsed Excitation
Remitted Intensity with Pulsed Excitation 1 0.9 0.8 Laser Pulse Raman Rayleigh Fluorescence
1 0.9

• 80 MHz repetition rate • 2 ns fluorescence lifetime • Rayleigh scattering ~t-3/2 • Raman scattering ~t-1/2

Normalized Power

0.7 0.6 0.5
Normalized Power

Remitted Intensity with Pulsed Excitation

0.8 0.7 0.6 0.5 0.4 0.3 0.2 Laser Pulse Raman Rayleigh Fluorescence 40 50 60

0.4 0.3 0.2 0.1 0 0 5 10 15 20 Time (ns)

0.1 25 0 0

30
10

35
20

40
30 Time (ps)

77

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

Frontier Investigations: Pulsed Excitation

78

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
79

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).
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