Biomedical Raman Spectroscopy

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): , 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. 78 years ago

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 Bangalore, India First photographed Raman spectra

The Raman Effect: Inelastic Scattering
hni h(ni-nR) 3 2 1 S1 S0 hni Inelastic Scattering Virtual Level Energy transferred from incident light to molecular vibrations Emitted light has decreased energy (i<R) Energy difference in energy Rayleigh Raman (inelastic) (elastic) Scattering Scattering

Some Vibrations in Benzene
Kekule Chubby Checker Breathing

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

Outline The Raman Effect Biomedical Raman Spectroscopy Instrumentation
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

For small distortions, polarizability is linearly proportional to the displacement Resultant dipole: Rayleigh Scattering Anti-Stokes Raman Stokes Raman

Photo-Molecular Interactions
Anti-Stokes Stokes Rayleigh Scattering 3 2 1 n1 2 1 n’1 Virtual Levels Energy E=hR 2 1 n0 Auto- IR Rayleigh Stokes Anti-Stokes NIR Fluorescence Absorption Scattering Raman Scattering Fluorescence

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

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  < s Strong Raman scatterers have distributed electron clouds C=C -bonds

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:

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 FWHM R= 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

Comparison to Infrared Absorption
(N)IR absorption directly interrogates molecular vibrations Requires change in dipole moment No symmetric stretches observed No diatomic activity Only observed in NIR and IR spectral regions High water absorption Broad spectral features Raman requires a change in polarizability with vibrational motion Occurs at all wavelengths Weak signal Sharp spectral features for molecular fingerprinting Complementary techniques Symmetric molecules with a center of inversion have vibrations which are either Raman or IR active, but not both (e.g. benzene) Molecules with no symmetry are active in both methods

Comparison to Infrared Absorption

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 ( ): 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

Spectroscopic Advantages of NIR Raman
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 350 mW 1 min 3 2 1 n1 n0 Energy Virtual Level Fluorescence Raman (inelastic) Scattering 100 200 500 1000 2 000 102 1 104 106 Molar extinction coefficient  (10-3M-1cm-1); H20 (cm-1) H2O HbO Visible 10-2 NIR Excitation Melanin Wavelength (nm)

Diagnostic Advantages of Raman Spectroscopy
Wavelength selection No biopsy required Directly measures molecules Small concentrations Chemical composition Morphological analysis Quantitative analysis In vivo diagnosis

Laser Sources for Raman Spectroscopy
Wavelength (nm) Ar+ 488.0, 514.5 Kr+ 530.9, 647.1 He: Ne 632.8 Ti: Al2O3 (cw) Diode (InGaAs) 785, 830 Nd:YAG 1064

In Vitro Experimental Raman System
CCD Collimating lenses Band-pass filter Notch filter Ti: sapphire laser Argon ion pump laser NIR excitation (830 nm) f/4 Spectrograph Dichroic beam-splitter Confocal pinhole 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 shutter bandpass filter Diode Laser holographic grating Raman probe notch filter CCD

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

Optical Fiber Probes Problems Fiber background Low signal collection
Distorts signal Adds shot-noise Low signal collection Raman effect is weak Tissue is highly diffusive Fiber background  NA2

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 Tissue Raman Fiber background Tissue Sample x c x c *  NA2 From McCreery RL “Raman Spectroscopy for Chemical Analysis,” 2000. Tissue Sample

Filter Transmission Region of Interest

Solution #1: Filtering Problems Fiber background Low signal collection
Distorts signal Adds shot-noise Low signal collection Raman effect is weak Tissue is highly diffusive Solutions Micro-optical filters Short-pass excitation filter Long-pass collection filter Optimize optical design Characterize distribution of Raman light in tissue Define optimal geometry Design collection optics

Excitation Light Diffusing Through Tissue
Monte Carlo 1 mm Experimental

Solution #2: Optical Design
Problems Fiber background Distorts signal Adds shot-noise Low signal collection Raman effect is weak Tissue is highly diffusive Solutions Micro-optical filters Short-pass excitation filter Long-pass collection filter Optimize optical design Characterize distribution of Raman light in tissue Define optimal geometry Design collection optics

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 collection fibers aluminum jacket excitation fiber
1 mm 1.75 mm collection fibers 0.70 long-pass filter tube metal sleeve aluminum jacket 0.55 excitation fiber short-pass filter rod 2 mm retaining sleeve ball lens Single Ring Probe has 15 Fibers Motz et al. Appl Opt 43: 52 (2004)

Calcified Aorta

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 T NC Media Fibrous Cap Lumen Atheroma Intima
Normal Mildly Atherosclerotic Plaque Ruptured Plaque Media Fibrous Cap Lumen T Atheroma Intima NC Adventitia T: thrombus NC: necrotic core 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

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 Raman Spectroscopy Quantitative biochemical information Morphological analysis

In Vitro Experimental Methods
Macroscopic Raman Spectroscopy 1 mm3 volumes of excised tissue are examined mW excitation with 830 nm laser light s collection times Comparison with histopathology for disease classification Principal Component Analysis

Raman Spectral Pathology of Atherosclerosis
Ca hydroxyapatite proteins calcified plaque cholesterol -carotene proteins lipid-rich plaque collagen elastin actin normal artery Image from medstat.med.utah.edu/WebPath/webpath.html

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

Morphological Model Hypothesis
Raman spectroscopy detects molecules (chemicals)  Every morphological feature has a distinct molecular structure  Each morphological feature has a unique Raman spectrum Chemical analysis of morphological structures Morphological modeling of coronary arteries

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

In Vitro Experimental Methods
Macroscopic Raman Spectroscopy 1 mm3 volumes of excised tissue are examined mW excitation with 830 nm laser light 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 s collection times Development of morphological model Spectroscopic mapping of tissue sections

Atherosclerosis In Vitro: Confocal Microscopy
Foam Cell Elastic Lamina 0.1 mm Smooth Muscle Cell Intima Media Adventitia ~(2x2x2) m3 Sampling Volume

Coronary Artery Morphological Structures
Buschman HPJ, et al. Cardiovascular Pathology 10(2), (2001)

Morphological Model of Coronary Arteries
Normal Coronary Artery Non-Calcified Plaque Calcified Plaque Residual Macroscopic Data Microscopic Model Fit Buschman HPJ, Motz JT, et al. Cardiovascular Pathology 10(2), (2001)

Morphological Assay of Coronary Arteries
Normal Coronary Artery Structure Contribution Collagen % Cholesterol % Calcification % Elastic Lamina % Fat % Foam Cell / Core % -Carotene % Smooth Muscle % Mildly Calcified Plaque Structure Contribution Collagen % Cholesterol % Calcification % Elastic Lamina % Fat % Foam Cell / Core % -Carotene % Smooth Muscle %

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), (2001)

Raman Morphometry of Coronary Artery
Buschman HPJ, et al. Cardiovascular Pathology 10(2), (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 Motz JT et al., J Biomed Opt 11(2):

Clinical Data: Atheromatous Plaque
1.8 mm deep calcification 0.4 mm 0.1 mm Motz JT et al., J Biomed Opt 11(2):

Clinical Data: Calcified Plaque
50 m Motz JT et al., J Biomed Opt 11(2):

Clinical Data: Ruptured Plaque
0.1 mm 0.4 mm Motz JT et al., J Biomed Opt 11(2):

Clinical Data: Thrombotic Plaque
0.4 mm 0.1 mm Motz JT et al., J Biomed Opt 11(2):

Clinical Data: Representative Analysis
Model Component Intimal Fibroplasia Atheromatous Plaque Calcified Plaque Ruptured Plaque Thrombotic Plaque Collagen (%) 9 7 Cholesterol (%) 44 2 27 14 Calcification (%) 16 71 1 12 Elastic Lamina (%) 4 3 Adventitial Fat (%) 50 13 Lipid Core (%) -Carotene (%) 23 Smooth Muscle (%) 28 47 61 Hemoglobin (a.u.)

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
Normal Breast Tissue Malignant Breast Tumor 100 mW excitation, 1 second collection

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

High-Wavenumber Raman Spectroscopy
Normal Bladder High-Wavenumber H&E lp: lamina propria u: urothelium DNA Cholesteryl palmitate Cholesteryl linoleate Triolein Collagen Actin Glycogen Koljenovic S et al., J Biomed Opt 10(3): (2005)

Frontier Investigations: Pulsed Excitation
80 MHz repetition rate 2 ns fluorescence lifetime Rayleigh scattering ~t-3/2 Raman scattering ~t-1/2 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: (2003).

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