Jason T. Motz

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.

Optical fiber probe for biomedical Raman spectroscopy

In vitro experiments have demonstrated the ability of Raman spectroscopy to diagnose a wide variety of diseases. Recent in vivo investigations performed with optical fiber probes were promising but generally limited to easily accessible organs, often requiring relatively long collection times. We have implemented an optical design strategy to utilize system throughput fully by characterizing the Raman distribution from tissue. This scheme optimizes collection efficiency, minimizes noise, and has resulted in small-diameter, highly efficient Raman probes that are capable of collecting high-quality data in 1 s. Performance has been tested through simulations and experiments with tissue models and several in vitro tissue types, demonstrating that this new design can advance Raman spectroscopy as a clinically practical technique.

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.

In vivo Raman spectral pathology of human atherosclerosis and vulnerable plaque.

The rupture of vulnerable atherosclerotic plaque accounts for the majority of clinically significant acute cardiovascular events. Because stability of these culprit lesions is directly related to chemical and morphological composition, Raman spectroscopy may be a useful technique for their study. Recent developments in optical fiber probe technology have allowed for the real-time in vivo Raman spectroscopic characterization of human atherosclerotic plaque demonstrated in this work. We spectroscopically examine 74 sites during carotid endarterectomy and femoral artery bypass surgeries. Of these, 34 are surgically biopsied and examined histologically. Excellent signal-to-noise ratio spectra are obtained in only 1 s and fit with an established model, demonstrating accurate tissue characterization. We also report the first evidence that Raman spectroscopy has the potential to identify vulnerable plaque, achieving a sensitivity and specificity of 79 and 85%, respectively. These initial findings indicate that Raman spectroscopy has the potential to be a clinically relevant diagnostic tool for studying cardiovascular disease.

Real-time Raman system for in vivo disease diagnosis

Raman spectroscopy has been well established as a powerful in vitro method for studying biological tissue and diagnosing disease. The recent development of efficient, high-throughput, low-background optical fiber Raman probes provides, for the first time, the opportunity to obtain real-time performance in the clinic. We present an instrument for in vivo tissue analysis which is capable of collecting and processing Raman spectra in less than 2 s. This is the first demonstration that data acquisition, analysis, and diagnostics can be performed in clinically relevant times. The instrument is designed to work with the new Raman probes and includes custom written LabVIEW and Matlab programs to provide accurate spectral calibration, analysis, and diagnosis along with important safety features related to laser exposure. The real-time capabilities of the system were demonstrated in vivo during femoral bypass and breast lumpectomy surgeries. Such a system will greatly facilitate the adoption of Raman spectroscopy into clinical research and practice.

Three-dimensional miniature endoscopy

A single optical fibre acts as a flexible probe to transmit a superior image of an internal landscape.The narrow viewMicroendoscopy, using devices a millimetre or so in diameter, is an accepted technique in ophthalmics, tumour diagnosis and other medical specialities. Submillimetre-diameter devices have been used in some clinical applications but have not been widely adopted because of their rigidity and poor image quality. A new type of endoscope has now been developed that can transmit video-rate, three-dimensional images from flexible probes that are the diameter of a single optical fibre, at 80–250 μm, comparable in size to a human hair.

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.

Model‐based biological Raman spectral imaging

Raman spectral imaging is a powerful tool for determining chemical information in a biological specimen. The challenge is to condense the large amount of spectral information into an easily visualized form with high information content. Researchers have applied a range of techniques, from peak‐height ratios to sophisticated models, to produce interpretable Raman images. The purpose of this article is to review some of the more common imaging approaches, in particular principal components analysis, multivariate curve resolution, and Euclidean distance, as well as to present a new technique, morphological modeling. How to best extract meaningful chemical information using each imaging approach will be discussed and examples of images produced with each will be shown. J. Cell. Biochem. Suppl. 39: 125–137, 2002.

Characterization of Atherosclerotic Plaques by Laser Speckle Imaging

Background—A method capable of determining atherosclerotic plaque composition and measuring plaque viscoelasticity can provide valuable insight into intrinsic features associated with plaque rupture and can enable the identification of high-risk lesions. In this article, we describe a new optical technique, laser speckle imaging (LSI), that measures an index of plaque viscoelasticity. We evaluate the potential of LSI for characterizing atherosclerotic plaque. Methods and Results—Time-varying helium-neon laser speckle images were acquired from 118 aortic plaque specimens from 14 human cadavers under static and deforming conditions (0 to 200 &mgr;m/s). Temporal fluctuations in the speckle patterns were quantified by exponential fitting of the normalized cross-correlation of sequential frames in each image series of speckle patterns to obtain the exponential decay time constant, &tgr;. The decorrelation time constants of thin-cap fibroatheromas (TCFA) (&tgr;=47.5±19.2 ms) were significantly lower than those of other atherosclerotic lesions (P<0.001), and the sensitivity and specificity of the LSI technique for identifying TCFAs were >90%. Speckle decorrelation time constants demonstrated strong correlation with histological measurements of plaque collagen (R=0.73, P<0.0001), fibrous cap thickness (R=0.87, P<0.0001), and necrotic core area (R=−0.81, P<0.0001). Under deforming conditions (10 to 200 &mgr;m/s), &tgr; correlated well with cap thickness in necrotic core fibroatheromas (P>0.05). Conclusions—The measurement of speckle decorrelation time constant from laser speckle images provides an index of plaque viscoelasticity and facilitates the characterization of plaque type. Our results demonstrate that LSI is a highly sensitive technique for characterizing plaque and identifying thin-cap fibroatheromas.

Spectral-domain spectrally-encoded endoscopy

Spectrally-encoded miniature endoscopy uses a single optical fiber and wavelength division multiplexing to obtain macroscopic images through miniature, flexible probes. In turn, it has the potential to enable two- and three-dimensional imaging within the body at locations that are currently difficult to access with conventional endoscopes. Here we present a novel detection scheme for spectrally-encoded endoscopy using spectral-domain interferometry. Compared to previous time-domain configurations, this new detection method results in greater than 1000-fold increase in sensitivity (77 dB), a 6-fold increase in imaging speed (30 volumes per second), and a 2-fold increase in depth range (2.8 mm). We demonstrate spectrally-encoded, spectral-domain detection by conducting video-rate, three-dimensional imaging in a variety of specimens, including the paws of a mouse embryo and excised human ear bones. Our results show that this new technology enables video rate spectrally-encoded endoscopy and will therefore be useful for a variety of minimally invasive medical applications.