The use of Raman spectroscopy to provide characterization and diagnosis of biological tissues has shown increasing
success in recent years. Most of this work has been performed using near-infrared laser sources such as 785 or 830 nm,
in a balance of reduced intrinsic fluorescence in the tissues and quantum efficiency in the silicon detectors often used.
However, even at these wavelengths, many tissues still exhibit strong or prohibitive fluorescence, and these wavelengths
still cause autofluorescence in many common sampling materials, such as glass. In this study, we demonstrate the use of
1064 nm dispersive Raman spectroscopy for the study of biological tissues. A number of tissues are evaluated using the
1064 nm system and compared with the spectra obtained from a 785 nm system. Sampling materials are similarly
compared. These results show that 1064 nm dispersive Raman spectroscopy provides a viable solution for measurement
of highly fluorescent biological tissues such as liver and kidney, which are difficult or impossible to extract Raman at
Shifted Excitation Raman Difference Spectroscopy (SERDS) implemented with two wavelength-stabilized laser diodes
with fixed wavelength separation is discussed as an effective method for dealing with the effects of fluorescence in
Raman spectroscopic analysis. In this presentation we discuss the results of both qualitative and quantitative SERDS
analysis of a variety of strongly fluorescing samples, including binary liquid mixtures. This application is enabled by the
Volume Bragg Grating® (VBG®) technology, which allows manufacturing of compact low-cost high-power laser
sources, suitable for extending the SERDS methodology to portable Raman spectrometers.
We report on a variety of BaySpec’s newly developed Raman spectrometers and microscopes combining multiple
excitation wavelengths and detection ranges. Among those there are the world’s first dual-wavelength near infrared
(NIR) and infrared miniature Raman spectral engines built with Volume Phase Gratings (VPGTM), and the world’s first
three-wavelength (532, 785, and 1064-nm) excitation Raman microscope. Having multiple wavelength excitations in one
unit offers extreme flexibility and convenience to identify the best laser wavelength and investigate a great variety of
real-world samples. In real-world Raman measurements, fluorescence is the biggest obstacle which significantly reduces
the quality of the Raman spectra. We demonstrate many examples spanning from explosives to street drugs to conclude
that for those samples, 1064-nm Raman is fluorescence-free and best suited for identification. Other types of
miniaturized Raman spectrometers have been realized, enabling handheld, portable, or at-line/ on-line applications for
real-world sample measurements, such as threat determination of explosives, chemical and biological materials, quality
assurance and contamination control for food safety, and forensics such as evidence gathering, narcotics identification,
We investigate the potential of near-infrared Raman microspectroscopy to differentiate between normal and malignant skin lesions. Thirty-nine skin tissue samples consisting of normal, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma from 39 patients were investigated. Raman spectra were recorded at the surface and at 20-µm intervals below the surface for each sample, down to a depth of at least 100 µm. Data reduction algorithms based on the nonlinear maximum representation and discrimination feature (MRDF) and discriminant algorithms using sparse multinomial logistic regression (SMLR) were developed for classification of the Raman spectra relative to histopathology. The tissue Raman spectra were classified into pathological states with a maximal overall sensitivity and specificity for disease of 100%. These results indicate the potential of using Raman microspectroscopy for skin cancer detection and provide a clear rationale for future clinical studies.
We explore imaging of tissue microstructures using autofluorescence and light scattering methods implemented through a hyperspectral microscope design. This system utilizes long working distance objectives that enable off-axis illumination of tissue thereby allowing for excitation at any optical wavelength without requiring change of optical elements within the microscope. Spectral and polarization elements are easily and rapidly incorporated to take
advantage of spectral variations of spectroscopic optical signatures for enhanced contrast. The collection efficiency has been maximized such that image acquisition may be acquired within very short exposure times, a key feature for transferring this technology to a clinical setting. Preliminary studies using human and animal tissues demonstrate the feasibility of this approach for real-time imaging of intact tissues as they would appear in the operating room.
Several studies have demonstrated Raman spectroscopy to be capable of tissue diagnosis with accuracy rivaling that of histopathologic analysis. This technique obtains biochemical-specific information noninvasively, and can eliminate the pain, time, and cost associated with biopsy and pathological analysis. Furthermore, when used in a confocal arrangement, Raman spectra can be obtained from localized regions of the tissue. Skin cancers are an ideal candidate for this emerging technology, due to their obvious accessibility and presentation at specific depths. However, most commercially available confocal Raman microspectrometers are large, rigid systems ill-suited for clinical application. We developed a bench-top confocal Raman microspectrometer using a portable external-cavity diode laser excitation source. This system was used to study several skin lesions in vitro. Results show the depth-resolved Raman spectra can diagnose in vitro skin lesions with 96% sensitivity, 88% specificity, and 86% pathological classification accuracy. Based on the success of this study, a portable Raman system with a handheld confocal microscope was developed for clinical application. Preliminary in vivo data show several distinct spectral differences between skin pathologies. Diagnostic algorithms are planned for this continuing study to assess the capability of Raman spectroscopy for clinical skin cancer diagnosis.
Raman spectroscopy has been shown to have the potential for providing differential diagnosis in the cervix with high sensitivity and specificity in previous in vitro and in vivo studies. A clinical study was designed at Vanderbilt University Medical Center to further evaluate the potential of near IR Raman spectroscopy for in vivo detection of squamous intra-epithelial neoplasia, a pre-cursor to cervical cancer, in a clinical setting. In this pilot in vivo clinical study, using a portable system, Raman spectra are collected using clinically feasible integration times during colposcopic evaluation. Multiple Raman spectra were acquired form colposcopically normal and abnormal sites prior to excision of tissue from patients with known abnormalities of the cervix. Measured Raman spectra were processed for nosie and background fluorescence using novel signal processing techniques. The resulting spectra were correlated with the corresponding histological diagnosis to determine empirical differences in spectra between different diagnostic categories. Using histology as the gold standard, multivariate statistical techniques were also used to develop discrimination algorithms with the hopes of developing this technique into a real time, non-invasive diagnostic tool.
With an overall survival rate of about 35 percent, ovarian cancer claims more than 13,000 women in the US each year. It is estimated that roughly 1 in 70 women will develop ovarian cancer. Current screening techniques are challenged due to cost-effectiveness, variable false-positive results, and the asymptomatic nature of the early stages of ovarian cancer. The predominant screening method for ovarian cancers is transvaginal sonography (TVS). TVS is fairly accomplished at ovarian cancer detection, however it is inefficient in distinguishing between benign and malignant masses. Accurate diagnosis of the ovarian tumor relies on exploratory laparotomy, thus increasing the cost and hazard of false- positive screening methods. Raman spectroscopy has been sued successfully as a diagnostic tool in several organ systems in vitro. These studies have shown that Raman spectroscopy can be used to provide diagnosis of subtle changes in tissue pathology with high accuracy. Based on this success, we have developed a Raman spectroscopic system for application in the ovary. Using this system, the Raman signatures of normal and various types of non-normal human ovarian tissues were characterized in vitro. Raman spectra are being analyzed, and empirical as well as multivariate discriminatory algorithms developed. Based on the result of this study, a strategy for in vivo trials will be planned.