Maryann Fitzmaurice

Prospects for in vivo Raman spectroscopy.

Raman spectroscopy is a potentially important clinical tool for real-time diagnosis of disease and in situ evaluation of living tissue. The purpose of this article is to review the biological and physical basis of Raman spectroscopy of tissue, to assess the current status of the field and to explore future directions. The principles of Raman spectroscopy and the molecular level information it provides are explained. An overview of the evolution of Raman spectroscopic techniques in biology and medicine, from early investigations using visible laser excitation to present-day technology based on near-infrared laser excitation and charge-coupled device array detection, is presented. State-of-the-art Raman spectrometer systems for research laboratory and clinical settings are described. Modern methods of multivariate spectral analysis for extracting diagnostic, chemical and morphological information are reviewed. Several in-depth applications are presented to illustrate the methods of collecting, processing and analysing data, as well as the range of medical applications under study. Finally, the issues to be addressed in implementing Raman spectroscopy in various clinical applications, as well as some long-term directions for future study, are discussed.

Detection of preinvasive cancer cells

More than 85% of all cancers originate in the epithelium that lines the internal surfaces of organs throughout the body. Although these are readily treatable provided they are diagnosed in one of the preinvasive stages, early lesions are often almost impossible to detect. Here we present a new optical-probe technique based on light-scattering spectroscopy that is able to detect precancerous and early cancerous changes in cell-rich epithelia.

Gastrointestinal tissue diagnosis by laser-induced fluorescence spectroscopy at endoscopy

An endoscope-compatible, optical fiber system has been developed which can be used to obtain laser-induced fluorescence spectra of mucosal abnormalities during endoscopy in real time. The results of our previous in vitro studies have suggested that laser-induced fluorescence tissue spectra are sufficiently unique that they can be used to accurately diagnose mucosal abnormalities in some systems. To test this hypothesis in vivo, laser-induced fluorescence spectra were obtained during colonoscopy from 31 colonic adenomas, 4 hyperplastic polyps, and 32 examples of normal mucosa in 20 patients. The resulting spectra could be used to correctly differentiate adenomas from normal colonic mucosa and hyperplastic polyps in 97% of the specimens studied with the resulting sensitivity, specificity, and positive predictive value of 100%, 97%, and 94%, respectively. These results, although preliminary in nature, suggest that laser-induced fluorescence spectra can be used in the recognition and differential diagnosis of mucosal abnormalities at endoscopy.

Histopathology of Human Coronary Atherosclerosis by Quantifying Its Chemical Composition With Raman Spectroscopy

BACKGROUND Lesion composition, rather than size or volume, determines whether an atherosclerotic plaque will progress, regress, or rupture, but current techniques cannot provide precise quantitative information about lesion composition. We have developed a technique to assess the pathological state of human coronary artery samples by quantifying their chemical composition with near-infrared Raman spectroscopy. METHODS AND RESULTS Coronary artery samples (n=165) obtained from explanted recipient hearts were illuminated with 830-nm infrared light. Raman spectra were collected from the tissue and processed to quantify the relative weights of cholesterol, cholesterol esters, triglycerides and phospholipids, and calcium salts in the examined artery location. The artery locations were then classified by a pathologist and grouped as either nonatherosclerotic tissue, noncalcified plaque, or calcified plaque. Nonatherosclerotic tissue, which included normal artery and intimal fibroplasia, contained an average of approximately 4+/-3% cholesterol, whereas noncalcified plaques had approximately 26+/-10% and calcified plaques approximately 19+/-10% cholesterol in the noncalcified regions. The average relative weight of calcium salts was 1+/-2% in noncalcified plaques and 41+/-21% in calcified plaques. To make this quantitative chemical information clinically useful, we developed a diagnostic algorithm, based on a first set of 97 samples, that demonstrated a strong correlation of the relative weights of cholesterol and calcium salts with histological diagnoses of the same locations. This algorithm was then prospectively tested on a second set of 68 samples. The algorithm correctly classified 64 of these new samples, thus demonstrating the accuracy and robustness of the method. CONCLUSIONS The pathological state of a given human coronary artery may be assessed by quantifying its chemical composition, which can be done rapidly with Raman spectroscopic techniques. When Raman spectra are obtained clinically via optical fibers, Raman spectroscopy may be useful in monitoring the progression and regression of atherosclerosis, predicting plaque rupture, and selecting proper therapeutic intervention.

Detection of dysplasia at colonoscopy using laser-induced fluorescence: a blinded study

BACKGROUND Laser-induced fluorescence spectroscopy has the potential to detect colonic dysplasia in vivo. However, previous studies have limited their analyses to multivariate regression techniques and unblinded retrospective evaluation. The purpose of this study was to develop a probability-based algorithm to detect colonic dysplasia using laser-induced fluorescence spectroscopy and to evaluate it in a blinded manner. METHODS Fluorescence spectra were collected from normal mucosa and colonic polyps during colonoscopy using 370 nm excitation. Tissue was classified as normal, hyperplastic, or adenomatous by histologic examination. Preliminary data was used to devise an algorithm to differentiate tissue type based on probability distributions of the fluorescence intensity at 460 nm and the ratio of the intensity at 680 nm to that at 600 nm. The algorithm was then tested in a blinded fashion. RESULTS The algorithm correctly determined the tissue type in 88% of cases, equal to the agreement of independent pathologists. Sensitivity, specificity, and positive predictive value for the detection of dysplasia was 90%, 95%, and 90%, respectively. CONCLUSIONS Dysplasia was detected in vivo using fluorescence spectroscopy and a probability-based algorithm. This method may form the basis for a new surveillance technique for patients with increased risk for dysplastic transformation.

Biochemical analysis and mapping of atherosclerotic human artery using FT-IR microspectroscopy

We report the application of FT-IR microspectroscopy for in situ spectroscopic characterization of molecular constituents of human atherosclerotic lesions. Since water content in tissue affects conformation-sensitive protein vibrational bands, tissue specimens were examined under moist conditions. In all measurements, vibrational bands from water were found to dominate the spectrum. By removing these water contributions, well resolved bands due to tissue components were readily observed. Utilizing the high sensitivity and good spatial resolution of IR microspectroscopy, spectra from a sample volume of 40 x 40 x 4 microns3 were collected using unstained cryostat sections mounted on a BaF2 flat in neutral isotonic saline. Microstructures were confirmed histologically by light microscopy in stained serial sections. In the spectrum of normal intima, major bands due to amide I (1656 cm-1), amide II (1556 cm-1), and CH bending (1457 cm-1) vibrations of the proteins collagen and elastin were observed. In the spectrum of the intima of noncalcified atherosclerotic plaque, major bands due to both proteins and lipids were observed. The lipid bands at 1734, 1468, 1171 and 1058 cm-1 were assigned to the C = O (ester) stretch, CH2 bend, C--O (ester) stretch and C--O stretch, respectively. At a more detailed level, bands specific to free cholesterol, and cholesterol esters were identified. A plot of the integrated intensity ratio of these bands to the protein amide II mode versus depth from the luminal surface confirmed a heterogeneous distribution of these constituents in the atheromatous core. In the spectra of calcified atherosclerotic plaque, bands were attributed to three types of biochemical microstructures: proteins (1657, 1555, 1243 cm-1), lipids (1735, 1466, 1170, 1085, 1055 cm-1) and calcium minerals such as hydroxyapatite (1094, 1040, 962 cm-1), and carbonated apatite (1463, 1412, 872 cm-1). The results demonstrate that IR microspectroscopy can be used for in situ characterization of molecular constituents in human unstained arterial sections. The molecular information obtained from these studies could be important in understanding the pathogenesis of atherosclerosis.

Raman Spectroscopy and Fluorescence Photon Migration for Breast Cancer Diagnosis and Imaging

We are developing optical methods based on near infra‐red Raman spectroscopy and fluorescence photon migration for diagnosis and localization of breast cancer. We demonstrate the ability of Raman spectroscopy to classify accurately normal, benign and malignant breast tissues, an important step in developing Raman spectroscopic needle probes as a tool for improving the accuracy of needle biopsy. We also show that photon migration imaging can be used to localize accurately small fluorescent objects imbedded in a thick turbid medium with realistic optical properties, thus demonstrating the potential of this technique for optical imaging.

Diagnosing breast cancer using diffuse reflectance spectroscopy and intrinsic fluorescence spectroscopy

Using diffuse reflectance spectroscopy and intrinsic fluorescence spectroscopy, we have developed an algorithm that successfully classifies normal breast tissue, fibrocystic change, fibroadenoma, and infiltrating ductal carcinoma in terms of physically meaningful parameters. We acquire 202 spectra from 104 sites in freshly excised breast biopsies from 17 patients within 30 min of surgical excision. The broadband diffuse reflectance and fluorescence spectra are collected via a portable clinical spectrometer and specially designed optical fiber probe. The diffuse reflectance spectra are fit using modified diffusion theory to extract absorption and scattering tissue parameters. Intrinsic fluorescence spectra are extracted from the combined fluorescence and diffuse reflectance spectra and analyzed using multivariate curve resolution. Spectroscopy results are compared to pathology diagnoses, and diagnostic algorithms are developed based on parameters obtained via logistic regression with cross-validation. The sensitivity, specificity, positive predictive value, negative predictive value, and overall diagnostic accuracy (total efficiency) of the algorithm are 100, 96, 69, 100, and 91%, respectively. All invasive breast cancer specimens are correctly diagnosed. The combination of diffuse reflectance spectroscopy and intrinsic fluorescence spectroscopy yields promising results for discrimination of breast cancer from benign breast lesions and warrants a prospective clinical study.

Raman microspectroscopy of human coronary atherosclerosis: Biochemical assessment of cellular and extracellular morphologic structures in situ

BACKGROUND We have previously shown that Raman spectroscopy can be used for chemical analysis of intact human coronary artery atherosclerotic lesions ex vivo without tissue homogenization or extraction. Here, we report the chemical analysis of individual cellular and extracellular components of atherosclerotic lesions in different stages of disease progression in situ using Raman microspectroscopy. METHODS Thirty-five coronary artery samples were taken from 16 explanted transplant recipient hearts, and thin sections were prepared. Using a high-resolution confocal Raman microspectrometer system with an 830-nm laser light, high signal-to-noise Raman spectra were obtained from the following morphologic structures: internal and external elastic lamina, collagen fibers, fat, foam cells, smooth muscle cells, necrotic core, beta-carotene, cholesterol crystals, and calcium mineralizations. Their Raman spectra were modeled by using a linear combination of basis Raman spectra from the major biochemicals present in arterial tissue, including collagen, elastin, actin, myosin, tropomyosin, cholesterol monohydrate, cholesterol linoleate, phosphatidyl choline, triolein, calcium hydroxyapatite, calcium carbonate, and beta-carotene. RESULTS The results show that the various morphologic structures have characteristic Raman spectra, which vary little from structure to structure and from artery to artery. The biochemical model described the spectrum of each morphologic structure quite well, indicating that the most essential biochemical components were included in the model. Furthermore, the biochemical composition of each structure, indicated by the fit contributions of the biochemical basis spectra of the morphologic structure spectrum, was very consistent. CONCLUSIONS The Raman spectra of various morphologic structures in normal and atherosclerotic coronary artery may be used as basis spectra in a linear combination model to analyze the morphologic composition of atherosclerotic coronary artery lesions.

A one-layer model of laser-induced fluorescence for diagnosis of disease in human tissue: applications to atherosclerosis

A general model of tissue fluorescence which can be used to both (1) determine chemical and physical properties of the tissue and (2) design an optimal algorithm for clinical diagnosis of tissue composition is described. The model is based on a picture of tissue as a single, optically thick layer, in which fluorophores and absorbing species are homogeneously distributed. As a specific example, the model is applied to the laser-induced fluorescence of normal and atherosclerotic human aorta using 476-nm excitation. Methods for determining the relevant attenuation and fluorescence lineshapes are detailed, and these lineshapes are used to apply the model to data from 148 samples. The model parameters are related to the concentrations of the major arterial chromosphores, structural proteins, hemoglobin and ceroid. In addition, the model parameters are used to derive diagnostic algorithms for the presence of atherosclerosis. Utilizing a binary classification scheme, the presence or absence of pathology was determined correctly in 88% of the cases.<<ETX>>