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Biomedical Raman Spectroscopy
Jason T. Motz
Harvard Medical School and The Wellman Center for PhotomedicineMassachusetts General Hospital
11 April 2006
2
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.
3
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
4
The Raman Effect: Inelastic Scatteringhi
h(i-R)
hi3
2
1
0 S1
3
2
1
0 S0
Ene
rgy
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
5
Some Vibrations in Benzene
BreathingChubby CheckerKekule
400 600 800 1000 1200 1400 1600 18000
1
2
3
4
5x 104
Raman Shift (cm-1)
Inte
nsi
ty (
CC
D C
ou
nts
)
6
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
7
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
8
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
p E
0 cos(2 )i iE E t
0 cos 2 Rq q t
9
Classical Raman Physics
• For small distortions, polarizability is linearly proportional to the displacement
• Resultant dipole:
0 0
0
0 0 cos 2 ip E E t
Rayleigh Scattering
Anti-Stokes RamanStokes Raman
0 0
0
1cos 2 cos 2
2 i R i RE q t tq
10
3
2
1
0 n1
Auto- IR Rayleigh Stokes Anti-Stokes NIRFluorescence Absorption Scattering Raman Scattering Fluorescence
Ene
rgy Virtual Levels
2
1
0 n0
2
1
0 n’1
Photo-Molecular Interactions
-2000 -1000 0 1000 20000
20
40
60
80
100
Raman Shift (cm-1)
Inte
nsity
Anti-StokesStokes
Rayleigh
Scattering
E=hR
11
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
0dq
4Rh
i RA kT
S i R
Ie
I
0I I dz
12
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
13
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
14
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:
1E
hc c
1 1R
i R
c E h
15
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
R
16
Raman Spectrum of Cholesterol
Hanlon et al. “Prospects for in vivo Raman spectroscopy,” Phys Med Biol 45: R1 (2000)
17
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
18
General Applications of Raman Spectroscopy
• Structural chemistry
• Solid state
• Analytical chemistry
• Applied materials analysis
• Process control
• Microspectroscopy/imaging
• Environmental monitoring
• Biomedical
21
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
22
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
23
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
24
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
25
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
26
• 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
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
Phenylalanine
Amide III
CH2 bend
Carotenoid
C=C
Ester linkage
Sterol ring
Phosphate stretch
Inte
nsi
ty (
a.u
.)
Raman Shift (cm-1)
350 mW
1 min
Spectroscopic Advantages of NIR Raman
100 200 500 1000 2 000
102
1
104
106
Mol
ar e
xtin
ctio
n c
oeff
icie
nt
(1
0-3M
-1cm
-1);
H
20 (
cm-1) H2O
HbO
HbO
Visible
10-2
NIR ExcitationMelanin
Wavelength (nm)
3
2
1
0 n1
3
2
1
0 n0
Ene
rgy
Virtual Level
Fluorescence Raman (inelastic)Scattering
27
Diagnostic Advantages of Raman Spectroscopy
• Wavelength selection
• No biopsy required
• Directly measures molecules– Small concentrations– Chemical composition– Morphological analysis
• Quantitative analysis
• In vivo diagnosis
28
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
29
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
30
In Vitro Experimental Raman SystemCCD
Collimating lenses
Band-p
ass
filt
er
Notch filter
Ti: sapphirelaser
Argon ion pump laser
NIR excitation (830 nm)
f/4Spectrograph
CCD
Dichroic beam-splitter
Confocalpinhole
Motorized translation stage
CCD Camera
31
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)
32
Clinical Raman Systems
CCD
830 nmbandpassfilter
notch filter
holographic grating
Ram
an p
robe
shutter
Diode Laser
33
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
34
Early in vivo Data
• Simple 6-around-1 optical fiber probe• 100 mW excitation, 3 second collection
35
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
36
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
x c
Tissue Sample
37
Filter Transmission
0 500 1000 15000
20
40
60
80
100
Raman Shift (cm-1)
Tra
nsm
issi
on
(%
)
Collection FilterExcitation Filter
Region of Interest
38
Problems
1. Fiber background• Distorts signal
• Adds shot-noise
2. Low signal collection• Raman effect is weak
• Tissue is highly diffusive
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
40
Problems
1. Fiber background• Distorts signal
• Adds shot-noise
2. Low signal collection• Raman effect is weak
• Tissue is highly diffusive
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
41
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
42
Raman Probe Design
2 mm
retainingsleeve
ball lens
1 m
m
1.75 mm
collection fibers
0.70
long-passfilter tube
metal sleeve
aluminum jacket
0.55
excitation fiber
short-passfilter rod
Single Ring Probe has 15 FibersMotz et al. Appl Opt 43: 52 (2004)
43
0 500 1000 15000
100
200
300
400
500
600
700
800
Raman Shift (cm-1)
Inte
nsity (
CC
D c
ounts
/mW
/s)
Single Ring, sapphire lensLab system
Calcified Aorta
44
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
45
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
46
Arterial Anatomy
Lumen
Intima
Adventitia
Media
TAtheroma
NC
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
47
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
48
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
49
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
50
Emerging Diagnostic Techniques
• Magnetic resonance imaging
• External ultrasound
• Positron emission tomography
• Electron beam computed tomography
• Thermography
• Elastography
• Intravascular ultrasound
• Optical coherence tomography
Non-Invasive
51
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
52
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
54
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+…
56
Potential Features for Spectral Identification
Collagen
Elastin
Actin
Adventitial fat
-carotene
Foam cells
Cholesterol
Necrotic core
Calcification
Hemoglobin
Fibrin
57
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
58
Atherosclerosis In Vitro: Confocal Microscopy
AdventitiaMediaIntima ~(2x2x2) m3 Sampling Volume
800 1000 1200 1400 1600 1800
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
Inte
nsity
(a.
u.)
Raman Shift (cm-1)
Elastic Lamina
800 1000 1200 1400 1600 1800-0.4
-0.2
0.0
0.2
0.4
0.6
0.8In
tens
ity (
a.u.
)
Raman Shift (cm-1)
Foam Cell
800 1000 1200 1400 1600 1800
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
Inte
nsity
(a.
u.)
Raman Shift (cm-1)
Smooth Muscle Cell
0.1 mm
59
Coronary Artery Morphological Structures
Buschman HPJ, et al. Cardiovascular Pathology 10(2), 69-82 (2001)
60
800 1200 1600
Inte
nsi
ty (a.
u.)
Raman shift (cm-1)
800 1200 1600
Inte
nsity
(a.u
.)
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
Inte
nsi
ty (a.
u.)
Raman shift (cm-1)
Normal Coronary Artery Non-Calcified Plaque
Morphological Model of Coronary Arteries
61
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
Inte
nsity
(a.
u.)
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
Inte
nsity
(a.
u.)
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
62
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
63
Coronary Artery Disease Classification:A Prospective Study
Buschman HPJ, Motz JT, et al. Cardiovascular Pathology 10(2), 59-68 (2001)
64
Raman Morphometry of Coronary Artery
Buschman HPJ, et al. Cardiovascular Pathology 10(2), 59-68 (2001)
65
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
66
Clinical Data: Intimal Fibroplasia (Normal)
0.4 mm
800 1000 1200 1400 1600 1800
-1
-0.5
0
0.5
1
Raman Shift (cm-1)
Inte
nsity
(a.
u.)
DataFitResidual
Motz JT et al., J Biomed Opt 11(2): 021003
67
Clinical Data: Atheromatous Plaque
0.4 mm
0.1 mm
800 1000 1200 1400 1600 1800
-0.5
0
0.5
1
Raman Shift (cm-1)
Inte
nsity
(a.
u.)
DataFitResidual
1.8 mm deepcalcification
Motz JT et al., J Biomed Opt 11(2): 021003
68
Clinical Data: Calcified Plaque
50 m
800 1000 1200 1400 1600 1800
-0.5
0
0.5
1
Raman Shift (cm-1)
Inte
nsity
(a.
u.)
DataFitResidual
Motz JT et al., J Biomed Opt 11(2): 021003
69
Clinical Data: Ruptured Plaque
0.4 mm
0.1 mm
800 1000 1200 1400 1600 1800
-1.5
-1
-0.5
0
0.5
1
Raman Shift (cm-1)
Inte
nsity
(a.
u.)
DataFitResidual
Motz JT et al., J Biomed Opt 11(2): 021003
70
Clinical Data: Thrombotic Plaque
0.4 mm
0.1 mm
800 1000 1200 1400 1600 1800
-1.5
-1
-0.5
0
0.5
1
Raman Shift (cm-1)
Inte
nsity
(a.
u.)
DataFitResidual
Motz JT et al., J Biomed Opt 11(2): 021003
71
Clinical Data: Representative Analysis
Model Component Intimal
Fibroplasia
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
72
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
73
Application To Other Diseases
100 mW excitation, 1 second collection
800 1000 1200 1400 1600 1800
-0.5
0
0.5
1
Raman Shift (cm-1)
Inte
snity
(a.
u.)
Data Fit Residual
Normal Breast Tissue
800 1000 1200 1400 1600 1800-1.5
-1
-0.5
0
0.5
1
Raman Shift (cm-1)In
tesn
ity (
a.u.
)
Data Fit Residual
Malignant Breast Tumor
74
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
75
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
76
High-Wavenumber Raman Spectroscopy
Koljenovic S et al., J Biomed Opt 10(3): 031116 (2005)
DNA
Cholesteryl palmitate
Cholesteryl linoleate
Triolein
Collagen
Actin
Glycogen
Normal Bladder
High-Wavenumber H&E
lp: lamina propriau: urothelium
77
Frontier Investigations: Pulsed Excitation
0 5 10 15 20 25 30 35 400
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Time (ns)
Nor
mal
ized
Pow
er
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
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Time (ps)
Nor
mal
ized
Pow
er
Remitted Intensity with Pulsed Excitation
Laser PulseRamanRayleighFluorescence
Decay kinetics based on work of Everall N et al., Appl Spectrosc 55(12): 1701 (2001)
79
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
80
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.
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