Imaging human epithelial properties with polarized light-scattering spectroscopy.

Biomedical imaging with light-scattering spectroscopy (LSS) is a novel optical technology developed to probe the structure of living epithelial cells in situ without need for tissue removal. LSS makes it possible to distinguish between single backscattering from epithelial-cell nuclei and multiply scattered light. The spectrum of the single backscattering component is further analyzed to provide quantitative information about the epithelial-cell nuclei such as nuclear size, degree of pleomorphism, degree of hyperchromasia and amount of chromatin. LSS imaging allows mapping these histological properties over wide areas of epithelial lining. Because nuclear enlargement, pleomorphism and hyperchromasia are principal features of nuclear atypia associated with precancerous and cancerous changes in virtually all epithelia, LSS imaging can be used to detect precancerous lesions in optically accessible organs.

Reversible molecular adsorption based on multiple-point interaction by shrinkable gels.

A general approach is presented for creating polymer gels that can recognize and capture a target molecule by multiple-point interaction and that can reversibly change their affinity to the target by more than one order of magnitude. The polymers consist of majority monomers that make the gel reversibly swell and shrink and minority monomers that constitute multiple-point adsorption centers for the target molecule. Multiple-point interaction is experimentally proven by power laws found between the affinity and the concentration of the adsorbing monomers within the gels.

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.

Characterization of the fluorescent morphological structures in human arterial wall using ultraviolet-excited microspectrofluorimetry.

In this study, the fluorescent morphological structures in normal coronary artery, normal aorta, and atherosclerotic aorta were histochemically identified and spectroscopically characterized in situ using ultraviolet-excited microspectrofluorimetry. Excitation wavelengths of 290 nm and 310/312 nm were employed to observe two distinct fluorescence bands, with peak emission wavelengths near 335 nm and 380 nm, respectively. Emission of the short wavelength 335 nm band, previously assigned to tryptophan residues in tryptophan-containing proteins, was observed from all the morphological structures in the vessel walls and was isolated in groups of smooth muscle cells in aorta and coronary artery media. The long wavelength 380 nm band was assigned to distinct fluorophores associated with the structural proteins collagen and elastin and was observed in collagen fibers and elastic fibers, respectively. The corresponding morphological structures in normal aorta, normal coronary artery, and atherosclerotic aorta exhibited similar fluorescence lineshapes. In atherosclerotic plaque, a distinct fluorescence band, peaking near 370 nm, was observed in the emission from both ceroid granules and necrotic core. Using a simple, quantitative model, differing contributions of collagen, elastin, and tryptophan-containing protein fluorescence were shown to account for over 95% of the emission from the intima, media, and adventitia layers of non-necrotic aorta and coronary artery.

Removal of surgically induced fibrous arterial plaques by argon ion laser angiosurgery using a multifiber delivery system. An experimental study in the dog.

Removal of intravascular atherosclerotic obstructions by laser irradiation has gained the attention of many investigators, but has proven to be considerably more difficult to accomplish than initially envisioned. We tested, in an animal model, an argon ion laser delivery system that permits control of (1) laser power, (2) exposure time, and (3) laser beam spot size. The study was conducted on surgically, induced focal fibrous plaques in the carotid arteries of nine dogs. Plaque removal, vessel patency, and healing were evaluated angiographically and by light and electron microscopy at intervals up to 60 days after treatment. Results showed that intravascular obstructions could be removed, healing occurred, and vessels remained patent for up to 60 days.

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.

Spectral pathology.

We are investigating the use of optical spectroscopy (fluorescence, reflectance, Raman scattering) for detecting precancerous lesions in the mucosal linings of hollow organs. We present a morphological model for extracting quantitative pathological information from fluorescence spectra, using colonic dysplasia as an example. The potential of this technique in providing histological information in real time without the need for tissue removal is discussed.

Feasibility of field-based light scattering spectroscopy.

Light scattering spectroscopy (LSS) is a new technique capable of accurately measuring the features of nuclei and other cellular organelles in situ. We present the considerations required to implement and interpret field-based detection in LSS, where the scattered electric field is detected interferometrically, and demonstrate that the technique is experimentally feasible. A theoretical formalism for modeling field-based LSS signals based on Mie scattering is presented. Phase-front uniformity is shown to play an important and novel role. Results of heterodyne experiments with polystyrene microspheres that localize LSS signals to a region about 30 microns in axial extent are reported. In addition, differences between field-based LSS and the earlier intensity-based LSS are discussed.

Optical computed tomography in a turbid medium using early arriving photons.

We employ photon migration to image absorbing objects embedded in a turbid medium. For improved resolution, we use early arriving photons (a few hundred picoseconds in excess of the time of flight), a regime in which the diffusion approximation breaks down. Our image reconstruction method is based on extension of x-ray computed tomography (CT) to the optical regime. The CT algorithm must be generalized to take into account the distributions of photon paths. We express the point spread function (PSF) in terms of the Green's function for the transport equation. This PSF then provides weighting functions for use in a generalized series expansion method of x-ray CT. Experiments were performed on a turbid medium with scattering and absorption properties similar to those of human breast tissue. Multiple absorbers were embedded into the medium to mimic tumors. Coaxial transmission scans were collected in two projections, and the early-time portions were analyzed. Through accurate modeling, we could remove the blurring associated with multiple scattering and obtain high-resolution images. Our results show that the diffusion approximation PSF is inadequate to describe the early arriving photons. A PSF incorporating causality is required to reconstruct accurate images of turbid media.

Laser-induced thermoelastic deformation: a three-dimensional solution and its application to the ablation of biological tissue.

Under certain conditions, laser light incident on a target material can induce an explosive removal of some material, a process called laser ablation. The photomechanical model of laser ablation asserts that this process is initiated when the laser-induced stresses exceed the strength of the material in question. Although one-dimensional calculations have shown that short pulsed lasers can create significant transient tensile stresses in target materials, the stresses last for only a few nanoseconds and the spatial location of the peak stresses is not consistent with experimental observations of material failure in biological tissues. Using the theory of elasticity, analytical expressions have been derived for the thermoelastic stresses and deformations in an axially symmetric three-dimensional solid body caused by the absorption of laser light. The full three-dimensional solution includes three stresses, radial, circumferential and shear, which are necessarily absent in the simple one-dimensional solution. These stresses have long-lived components that exist for eight orders of magnitude longer in time than the acoustic transients, an important point when the details of dynamic fracture are considered. Many important qualitative features are revealed including the spatial location of the peak stresses, which is more consistent with experimental observations of failure.

Instrumentation for multi-modal spectroscopic diagnosis of epithelial dysplasia.

Reflectance and fluorescence spectroscopies have shown great promise for early detection of epithelial dysplasia. We have developed a clinical reflectance spectrofluorimeter for multimodal spectroscopic diagnosis of epithelial dysplasia. This clinical instrument, the FastEEM, collects white light reflectance and fluorescence excitation-emission matrices (EEM's) within a fraction of a second. In this paper we describe the FastEEM instrumentation, designed for collection of multi-modal spectroscopic data. We illustrate its performance using tissue phantoms with well defined optical properties and biochemicals of known fluorescence properties. In addition, we discuss our plans to develop a system that combines a multi-spectral imaging device for wide area surveillance with this contact probe device.

Reagentless blood analysis by near-infrared Raman spectroscopy.

BACKGROUND: Raman spectroscopy has advantages over infrared absorption spectroscopy. Combined with a novel multivariate technique, hybrid linear analysis (HLA), low prediction error is expected. METHODS: A near-infrared (NIR) light source excited Raman signals, and a charge coupled device (CCD) camera was used to collect the signal. Samples were collected from 69 individuals for 7 weeks. The standard multivariate calibration technique, partial least squares (PLS) and HLA were both used to analyze the collected spectra. A Clarke error grid was used to evaluate the usefulness of the glucose measurement in serum. RESULTS: The root mean squared error of prediction (RMSEP) for glucose in serum obtained with PLS is 21 mg/dL, and the RMSEP obtained with HLA is 17 mg/dL. In whole blood, the PLS RMSEP for glucose was 79 mg/dL, and HLA predictions had an RMSEP of 63 mg/dL. CONCLUSIONS: The measurement technique was robust over the 7-week period. HLA was shown to generate a lower prediction error than PLS. The predictions by both PLS and HLA were clinically acceptable. The result with whole blood requires further improvement.

Spectroscopic diagnosis of colonic dysplasia.

We have developed a method for defining diagnostic algorithms for pathologic conditions based on fluorescence spectroscopy. We apply this method to human colon tissue and show that fluorescence can be used to diagnose the presence or absence of colonic adenoma. This method uses fluorescence excitation-emission matrices (EEM) to identify optimal excitation regions for obtaining fluorescence emission spectra which can be used to differentiate normal and pathologic tissues. In the case of normal and adenomatous colon tissue, these were found to be: 330, 370, and 430 nm +/- 10 nm. At these excitation wavelengths, emission wavelengths for use in diagnostic algorithms are identified from average difference and ratio of the spectra from normal and pathologic tissues. In colon tissue, at 370 nm excitation, 404, 480, and 680 nm were found to be useful emission wavelengths for diagnosing the presence of adenoma in vitro. The basis of colon tissue autofluorescence was investigated using EEM of pure molecules and relevant excitation-emission maxima in the literature.

Raman spectroscopy and fluorescence photon migration for breast cancer diagnosis and imaging.

We are developing optical methods based on near infrared 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.

Near-infrared fluorescence spectroscopy detects Alzheimer's disease in vitro.

The purpose of this study was to investigate whether near-infrared (NIR) fluorescence spectroscopy could be used to detect Alzheimer's disease (AD) by brain tissue autofluorescence. Unfixed temporal cortex specimens from AD cases and age-matched, non-AD controls were frozen at autopsy and then thawed just prior to spectral measurement. Spectra of intrinsic tissue fluorescence induced by 647 nm light were recorded from 650 to 850 nm. We used principal component analysis of the tissue spectra from 17 AD cases and 5 non-AD control cases in a calibration study to establish a diagnostic algorithm. Retrospectively applied to the calibration set, the algorithm correctly classified 23 of 24 specimens. In a prospective study of 19 specimens from 5 AD brains and 2 non-AD control brains, 3 of the 4 control specimens and all AD specimens were correctly diagnosed. Both the excitation light used and the measured brain tissue autofluorescence are at NIR wavelengths that can propagate through skull and overlying tissue. Therefore, our results demonstrate an optical spectroscopic technique that carries direct molecular level information about disease. This is the first step toward a clinical tool that has the potential to be applied to the noninvasive diagnosis of AD in living patients.

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.

Diagnosis of head and neck precancerous lesions in an animal model using fluorescence spectroscopy.

Laser-induced fluorescence (LIF) of tissues depends on their biochemical and histomorphologic characteristics. LIF spectroscopic properties of 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced precancerous and early cancerous lesions in a hamster buccal pouch mucosa model were studied. Fluorescence spectra from neoplastic lesions showed a characteristic fluorescence peak in the red region of the visible spectrum centered between 630 and 640 nm when excited with 410-nm light. Using this as a diagnostic criterion, 45 of 49 lesions studied were correctly diagnosed, including early dysplastic lesions. Follow-up study of four dysplastic lesions over 2 weeks revealed an increase in red fluorescence intensity. The findings of these experiments suggest that LIF spectroscopy may be a valuable noninvasive technique not only for early diagnosis of head and neck cancer, but also to probe a possible biochemical surrogate biomarker in the follow-up of suspected lesions.

Early diagnosis of upper aerodigestive tract cancer by autofluorescence.

OBJECTIVE: To explore the potential of autofluorescence spectroscopy as a tool for early detection of upper aerodigestive tract cancer. DESIGN: Autofluorescence spectral characteristics of 19 untreated oral and oropharyngeal lesions in 13 patients were studied with excitation wavelengths of 370 and 410 nm generated by a nitrogen pumped dye laser. Ten healthy volunteers were recruited to characterize the fluorescence spectra of normal mucosa at different oral sites and to study individual variations. Fluorescence intensity and line shape of the spectra from lesions were compared with the same parameters from the contralateral control site in the same individual. SETTING: Otolaryngology Research Center, Department of Otolaryngology-Head and Neck Surgery, New England Medical Center, Boston, Mass. RESULTS: The ratio of peak fluorescence intensities of the neoplastic lesions to contralateral normal control mucosa were consistently different compared with these ratios in benign lesions or normal mucosa. These differences were seen in 2 distinct regions of the fluorescence spectrum with both of the excitation wavelengths, but were more obvious with the excitation wavelength of 410 nm. Using these differences, we were able to correctly diagnose 17 of the 19 lesions studied, with 2 false-positive results. CONCLUSIONS: Neoplastic oral mucosa shows consistent differences in autofluorescence spectral intensity and line shape when compared with the normal mucosa in the same individual. These early results show that fluorescence spectroscopy may represent a useful technique for noninvasive early diagnosis of cancer of the upper aerodigestive tract.