Geurt Deinum

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.

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.

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.

Diagnosis of human coronary atherosclerosis by morphology-based Raman spectroscopy.

BACKGROUND Recent studies have shown that chemical composition and morphology, rather than anatomy (degree of stenosis), determine atherosclerotic plaque instability and predict disease progression. Current clinical diagnostic techniques provide accurate assessment of plaque anatomy, but have limited capability to assess plaque morphology in vivo. Here we describe a technique for a morphology-based diagnosis of atherosclerosis in the coronary arteries using Raman spectroscopy that can potentially be performed in vivo using optical fiber technology. METHODS Raman tissue spectra were collected from normal and atherosclerotic coronary artery samples in different stages of disease progression (n=165) from explanted transplant recipient hearts (n=16). Raman spectra from the elastic laminae (EL), collagen fibers (CF), smooth muscle cells (SMC), adventitial adipocytes (AA) or fat cells, foam cells (FC), necrotic core (NC), cholesterol crystals (CC), beta-carotene containing crystals (beta-C), and calcium mineralizations (CM) were used as basis spectra in a linear least squares-minimization (LSM) model to calculate the contribution of these morphologic structures to the coronary artery tissue spectra. RESULTS We developed a diagnostic algorithm that used the fit-contributions of the various morphologic structures to classify 97 coronary artery samples in an initial calibration data set as either nonatherosclerotic, calcified plaque, or noncalcified atheromatous plaque. The algorithm was subsequently tested prospectively in a second validation data set, and correctly classified 64 (94%) of 68 coronary artery samples. CONCLUSIONS Raman spectroscopy provides information about the morphologic composition of intact human coronary artery without the need for excision and microscopic examination. In the future, it may be possible to use this technique to analyze the morphologic composition of atherosclerotic coronary artery lesions and assess plaque instability and disease progression in vivo.

Histological Classification of Raman Spectra of Human Coronary Artery Atherosclerosis Using Principal Component Analysis

We present a nonparametric method of analysis of Raman spectra of coronary artery tissue to classify atherosclerotic lesions. The method correlates the principal component scores of the Raman spectra with the tissue pathology. A data set composed of 97 samples of human coronary artery was used to develop the diagnostic algorithm, and a second data set composed of 68 samples was then used to test this algorithm prospectively. The results show that the algorithm can accurately classify coronary artery tissue into three classes: nonatherosclerotic, noncalcified plaque, and calcified plaque. The accuracy of this classification scheme is comparable to that previously achieved by means of a biochemical analysis of the Raman spectra using the same data.

CO PHOTOLYSIS OF CYTOCHROME OXIDASE INVESTIGATED BY PS RESONANCE RAMAN SPECTROSCOPY

Low-power picosecond resonance Raman spectroscopy was used to investigate the identity of the axial ligand of heme a3 and relaxation processes in the heme a3 pocket of cytochrome oxidase after CO photolysis. Our results show that the proximal histidine remains ligated to heme a3 after CO photolysis excluding the transient ligation of a photolabile, endogenous ligand. Furthermore, the relaxation of the heme a3 macrocycle modes occurs on the sub ps time scale, while relaxation of the heme pocket to its equilibrium conformation takes place on the μs time scale.

Ligand Dynamics in the Binuclear Site in Cytochrome Oxidase

The dioxygen-reduction mechanism in cytochrome oxidase relies on proton control of the electron-transfer events that drive the process. Recent work on proton delivery and efflux channels in the protein that are relevant to substrate reduction and proton pumping is considered, and the current status of this area is summarized. Carbon monoxide photodissociation and the ligand dynamics that occur subsequent to photolysis have been valuable tools in probing possible coupling schemes for linking exergonic electron-transfer chemistry to endergonic proton translocation. Our picosecond-time-resolved Raman results show that the heme a 3-proximal histidine bond remains intact following CO photodissociation but that the local environment around the heme a 3 center in the photoproduct is in a nonequilibrium state. This photoproduct relaxes to its equilibrium configuration on the same time scale as ligand release occurs from CuB, which suggests a coupling between the two events and a potential signaling pathway between the site of O2 binding and reduction and the putative element, CuB, that links the redox chemistry to the proton pump.