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Infrared neural stimulation (INS) is a promising neurostimulation technique that can activate neural tissue with high spatial precision and without the need for exogenous agents. However, little is understood about how infrared light interacts with neural tissue on a cellular level, particularly within the living brain. In this study, we use calcium sensitive dye imaging on macroscopic and microscopic scales to explore the spatiotemporal effects of INS on cortical calcium dynamics. The INS-evoked calcium signal that was observed exhibited a fast and slow component suggesting activation of multiple cellular mechanisms. The slow component of the evoked signal exhibited wave-like properties suggesting network activation, and was verified to originate from astrocytes through pharmacology and 2-photon imaging. We also provide evidence that the fast calcium signal may have been evoked through modulation of glutamate transients. This study demonstrates that pulsed infrared light can induce intracellular calcium modulations in both astrocytes and neurons, providing new insights into the mechanisms of action of INS in the brain., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The overexpression of human epidermal growth factor receptor 2 (HER2) is associated with increased breast cancer recurrence and worse prognosis. Effective treatments such as trastuzumab and lapatinib for patients with HER2 overexpression target the blockade of HER2 signaling activities but are often limited by the emergence of acquired drug resistance. This study applied Raman spectroscopy to differentially identify the amplification status of HER2 in cells and to characterize the biochemical composition of lapatinib resistant and sensitive HER2+ breast cancer cells in response to the drug. Raman spectra from BT474 (HER2+ breast cancer cell), MCF-10A (HER2- control), and HER2+ MCF-10A (HER2+ control) were analyzed using lasso and elastic-net regularized generalized linear models (glmnet) for multivariate statistical analysis and were discriminated to groups of different HER2 expression status with an overall 99% sensitivity and specificity. Enhanced lipid content and decreased proteome were observed in HER2+ cells. With lapatinib treatment, lapatinib-resistant breast cancer cells demonstrated sustained lipogenesis compared with the sensitive cells.

Bone is the most common site for breast cancer metastases. One of the major complications of bone metastasis is pathological bone fracture caused by chronic bone loss and degeneration. Current guidelines for the prediction of pathological fracture mainly rely on radiographs or computed tomography, which are limited in their ability to predict fracture risk. The present study explored the feasibility of using Raman spectroscopy to estimate pathological fracture risk by characterizing the alterations in the compositional properties of metastatic bones. Tibiae with evident bone destruction were investigated using Raman spectroscopy. The carbonation level calculated by the ratio of carbonate/phosphate ν1 significantly increased in the tumor-bearing bone at all the sampling regions at the proximal metaphysis and diaphysis, while tumor-induced elevation in mineralization and crystallinity was more pronounced in the metaphysis. Furthermore, the increased carbonation level is positively correlated to bone lesion size, indicating that this parameter could serve as a unique spectral marker for tumor progression and bone loss. With the promising advances in the development of spatially offset Raman spectroscopy for deep tissue measurement, this spectral marker can potentially be used for future noninvasive evaluation of metastatic bone and prediction of pathological fracture risk.

Background: Inadvertent removal of parathyroid glands is a challenge in endocrine operations. There is a critical need for a diagnostic tool that provides sensitive, real-time parathyroid detection during procedures. We have developed an intraoperative technique using near-infrared (NIR) fluorescence for in vivo, real-time detection of the parathyroid regardless of its pathologic state., Methods: NIR fluorescence was measured intraoperatively from 45 patients undergoing parathyroidectomy and thyroidectomy. Spectra were measured from the parathyroid and surrounding neck tissues during the operation with the use of a portable, probe-based fluorescence system at 785-nm excitation. Accuracy was evaluated by comparison with histology or visual recognition by the surgeon., Results: NIR fluorescence detected the parathyroid in 100% of patients. Parathyroid fluorescence was stronger (1.2-18 times) than that of the thyroid with peak fluorescence at 822 nm. Surrounding tissues showed no auto-fluorescence. Disease state did not affect the ability to discriminate parathyroid glands but may account for signal variability., Conclusion: NIR fluorescence spectroscopy can detect intraoperatively the parathyroid regardless of tissue pathology. The signal may be caused by calcium-sensing receptors present in the parathyroid. The signal strength and consistency indicates the simplicity and effectiveness of this method. Its implementation may limit operative time, decrease costs, and improve operative success rates., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Background and Objectives: With the increasing use of fluorescence in medical applications, a comprehensive understanding of the effect of temperature on tissue autofluorescence is essential. The purpose of this study is to explore the effect of temperature on the fluorescence of porcine cornea and rat skin and determine the relative contributions of irreversible changes in optical properties and in fluorescence yield., Study Design/materials and Methods: Fluorescence, diffuse reflectance, and temperature measurements were acquired from excised porcine cornea and rat skin over a temperature range of 0-80 °C. A dual excitation system was used with a 337 nm pulsed nitrogen laser for the fluorescence and a white light source for the diffuse reflectance measurements. A thermal camera measured tissue temperature. Optical property changes were inferred from diffuse reflectance measurements. The reversibility of the change in fluorescence was examined by acquiring measurements while the tissue sample cooled from the highest induced temperature to room temperature., Results: The fluorescence intensity decreased with increasing tissue temperature. This fluorescence change was reversible when the tissue was heated to a temperature of 45 °C, but irreversible when heated to a temperature of 80 °C., Conclusion: Auto-fluorescence intensity dependence on temperature appears to be a combination of temperature-induced optical property changes and reduced fluorescence quantum yield due to changes in collagen structure. Temperature-induced changes in measured fluorescence must be taken into consideration in applications where fluorescence is used to diagnose disease or guide therapy., (Copyright © 2012 Wiley Periodicals, Inc.)

The goal of this research is to determine whether Raman spectroscopy (RS), an optical method that probes the vibrational modes of tissue components, can be used in vivo to study changes in the mouse cervix during pregnancy. If successful, such a tool could be used to detect cervical changes due to pregnancy, both normal and abnormal, in animal models and humans. For this study, Raman spectra were acquired before, during and after a 19-day mouse gestational period. In some cases, after Raman data was obtained, cervices were excised for structural testing and histological staining for collagen and smooth muscle. Various peaks of the Raman spectra, such as the areas corresponding to fatty acid content and collagen organization, changed as the cervix became softer in preparation for labor and delivery. These findings correspond to the increase in compliance of the tissue and the collagen disorganization visualized with the histological staining. The results of this study suggest that non-invasive RS can be used to study cervical changes during pregnancy, labor and delivery and can possibly predict preterm delivery before overt clinical manifestations, potentially lead to more effective preventive and therapeutic interventions.

Background and Objective: Laser preconditioning augments incisional wound healing by reducing scar tissue and increasing maximum tensile load of the healed wound [Wilmink et al. (2009) J Invest Dermatol 129(1): 205-216]. Recent studies have optimized treatments or confirmed results using HSP70 as a biomarker. Under the hypothesis that HSP70 plays a role in reported results and to better understand the downstream effects of laser preconditioning, this study utilized a probe-based Raman spectroscopy (RS) system to achieve an in vivo, spatio-temporal biochemical profile of murine skin incisional wounds as a function of laser preconditioning and the presence of HSP70., Study Design/materials and Methods: A total of 19 wild-type (WT) and HSP70 knockout (HSP70-/-) C57BL/6 mice underwent normal and laser preconditioned incisional wounds. Laser thermal preconditioning was conducted via previously established protocol (λ = 1.85 µm, H(0 ) = 7.64 mJ/cm(2) per pulse, spot diameter = 5 mm, Rep. rate = 50 Hz, τ(p) = 2 milliseconds, exposure time = 10 minutes) with an Aculight Renoir diode laser, with tissue temperature confirmed by real-time infrared camera measurements. Wound-healing progression was quantified by daily collection of a spatial distribution of Raman spectra. The results of RS findings were then qualified using standard histology and polarization microscopy., Results: Raman spectra yielded significant differences (t-test; α = 0.05) in several known biochemical peaks between WT and HSP70 (-/-) mice on wounds and in adjacent tissue early in the wound-healing process. Analysis of peak ratios implied (i) an increase in protein configuration in and surrounding the wound in WT mice, and (ii) an increased cellular trend in WT mice that was prolonged due to laser treatment. Polarization microscopy confirmed that laser treated WT mice showed increased heterogeneity in collagen orientation., Conclusions: The data herein supports the theory that HSP70 is involved in normal skin protein configuration and the cellularity of early wound healing. Laser preconditioning extends cellular trends in the presence of HSP70. Despite study limitations, RS provided a non-invasive method for quantifying temporal trends in altered wound healing, narrowing candidates and design for future studies with clinically applicable instrumentation., (Copyright © 2012 Wiley Periodicals, Inc.)

Many groups have used Raman spectroscopy for diagnosing cervical dysplasia; however, there have been few studies looking at the effect of normal physiological variations on Raman spectra. We assess four patient variables that may affect normal Raman spectra: Race/ethnicity, body mass index (BMI), parity, and socioeconomic status. Raman spectra were acquired from a diverse population of 75 patients undergoing routine screening for cervical dysplasia. Classification of Raman spectra from patients with a normal cervix is performed using sparse multinomial logistic regression (SMLR) to determine if any of these variables has a significant effect. Results suggest that BMI and parity have the greatest impact, whereas race/ethnicity and socioeconomic status have a limited effect. Incorporating BMI and obstetric history into classification algorithms may increase sensitivity and specificity rates of disease classification using Raman spectroscopy. Studies are underway to assess the effect of these variables on disease.

The risk of local recurrence for breast cancers is strongly correlated with the presence of a tumor within 1 to 2 mm of the surgical margin on the excised specimen. Previous experimental and theoretical results suggest that spatially offset Raman spectroscopy (SORS) holds much promise for intraoperative margin analysis. Based on simulation predictions for signal-to-noise ratio differences among varying spatial offsets, a SORS probe with multiple source-detector offsets was designed and tested. It was then employed to acquire spectra from 35 frozen-thawed breast tissue samples in vitro. Spectra from each detector ring were averaged to create a composite spectrum with biochemical information covering the entire range from the tissue surface to ∼2 mm below the surface, and a probabilistic classification scheme was used to classify these composite spectra as "negative" or "positive" margins. This discrimination was performed with 95% sensitivity and 100% specificity, or with 100% positive predictive value and 94% negative predictive value.

A major challenge in endocrine surgery is the intraoperative detection of parathyroid glands during both thyroidectomies and parathyroidectomies. Current localization techniques such as ultrasound and sestamibi scan are mostly preoperative and rely on an abnormal parathyroid for its detection. In this paper, we present near-infrared (NIR) autofluorescence as a nonintrusive, real-time, automated in vivo method for the detection of the parathyroid gland. A pilot in vivo study was conducted to assess the ability of NIR fluorescence to identify parathyroid glands during thyroid and parathyroidectomies. Fluorescence measurements at 785 nm excitation were obtained intra-operatively from the different tissues exposed in the neck region in 21 patients undergoing endocrine surgery. The fluorescence intensity of the parathyroid gland was found to be consistently greater than that of the thyroid and all other tissues in the neck of all patients. In particular, parathyroid fluorescence was two to eleven times higher than that of the thyroid tissues with peak fluorescence occurring at 820 to 830 nm. These results indicate that NIR fluorescence has the potential to be an excellent optical tool to locate parathyroid tissue during surgery.

Background and Objective: The current standard for diagnosis of skin cancers is visual inspection followed by biopsy and histopathology. This process can be invasive, subjective, time consuming, and costly. Optical techniques, including Optical Coherence Tomography (OCT) and Raman Spectroscopy (RS), have been developed to perform non-invasive characterization of skin lesions based on either morphological or biochemical features of disease. The objective of this work is to report a clinical instrument capable of both morphological and biochemical characterization of skin cancers with RS-OCT., Materials and Methods: The portable instrument utilizes independent 785 nm RS and 1,310 nm OCT system backbones. The two modalities are integrated in a 4″ (H) × 5″(W) × 8″(L) clinical probe. The probe enables sequential acquisition of co-registered OCT and RS data sets. The axial response of the RS collection in the skin was estimated using scattering phantoms. In addition, RS-OCT data from patients with cancerous and non-cancerous lesions are reported., Results: The RS-OCT instrument is capable of screening areas as large as 15 mm (transverse) by 2.4 mm (in depth) at up to 8 frames/second with OCT, and identifying locations to perform RS. RS signal is collected from a 44 µm transverse spot through a depth of approximately 530 µm. RS-OCT data sets from a superficial scar and a nodular BCC are reported to demonstrate the clinical potential of the instrument., Conclusion: The RS-OCT instrument reported here is capable of morphological and biochemical characterization of cancerous skin lesions in a clinical setting. OCT can visualize microstructural irregularities and perform an initial morphological analysis of the lesion. The images can be used to guide acquisition of biochemically specific Raman spectra. The two data sets can then be evaluated with respect to one another to take advantage of the mutually complimentary nature of RS and OCT., (Copyright © 2011 Wiley-Liss, Inc.)

Background and Objective: Previous studies demonstrated a decrease in fluorescence intensity as tissue temperature increased. In vitro samples were increased from room temperature and in vivo canine liver from body temperature. This study investigated variations in fluorescence intensity with temperatures starting at 14°C and compared in vivo and in vitro results for consistency., Study Design/material and Methods: A fiber optic-based noninvasive system was used to characterize the temperature effect on tissue fluorescence in hamster dorsal skin in vivo, and in sclera and cornea of enucleated pig eyes in vitro. As tissue was allowed to progress through the temperature range of 14-42°C, the spectra of auto-fluorescence with respect to temperature was sampled every 1-2 minutes. A pulsed nitrogen laser was used to excite fluorescence through a fiber optic probe with a source-detector aperture separation of 370 µm., Results: Fluorescence intensity decreased as temperature increased from 14 to 42°C in a phantom containing Rhodamine B dye. Results from both in vivo and in vitro tissue followed the same trend of decreasing intensity as tissue temperature increased from 14°C. Spectral intensity lineshape changed around 450 nm due to absorption from tissue., Conclusion: Cooling a tissue increased fluorescence intensity of skin in vivo, in all experiments. In vitro results were consistent with in vivo measurements., (Copyright © 2011 Wiley Periodicals, Inc.)

Raman spectroscopy (RS) and optical coherence tomography (OCT) are powerful tools for optical analysis of tissues with mutually complementary strengths and limitations. OCT excels at visualizing tissue microstructure but lacks molecular specificity, while RS can relay tissue biochemical composition but typically cannot relate microstructure. Previous implementations of combined RS-OCT have utilized a common sample arm while maintaining independent RS and OCT detection arms. We present the design and application of an integrated RS-OCT instrument with a common detection arm for both RS and OCT. The detector is a spectrograph capable of sequential detection of the 855-nm OCT signal and the Raman scatter generated by a 785-nm source. The capabilities of the instrument are demonstrated ex vivo in the calvaria and retina of rodents, as well as in vivo in human skin.

Transforming growth factor β (TGF-β) is an abundant bone matrix protein that influences osteoblast and osteoclast interactions to control bone remodeling. As such, TGF-β represents an obvious pharmacologic target with the potential to regulate both bone formation and resorption to improve bone volume and strength. To investigate the skeletal effect of TGF-β inhibition in vivo, we used an antibody (1D11) specifically directed at all three isoforms of TGF-β. Normal mice were treated with 1D11 or control antibody (4 weeks), and cortical and trabecular bone was assessed by micro-computed tomographic (µCT) scanning. Bone volume and cellular distribution were determined by histomorphometric analysis of vertebrae and long bones. Also, whole-bone strength was assessed biomechanically by three-point bend testing, and tissue-level modulus and composition were analyzed by nanoindentation and Raman microspectroscopy, respectively. TGF-β blockade by 1D11 increased bone mineral density (BMD), trabecular thickness, and bone volume by up to 54%, accompanied by elevated osteoblast numbers and decreased osteoclasts. Biomechanical properties of bone also were enhanced significantly by 1D11 treatment, with increased bending strength and tissue-level modulus. In addition, Raman microspectroscopy demonstrated that 1D11-mediated TGF-β inhibition in the bone environment led to an 11% increase in the mineral-to-collagen ratio of trabecular bone. Together these studies demonstrate that neutralizing TGF-β with 1D11 increases osteoblast numbers while simultaneously decreasing active osteoclasts in the marrow, resulting in a profound increase in bone volume and quality, similar to that seen in parathyroid hormone (PTH)-treated rodent studies., (© 2010 American Society for Bone and Mineral Research.)

We have previously demonstrated the discrimination of two layers of soft tissue, specifically normal breast tissue overlying breast tumor, using spatially offset Raman spectroscopy (SORS). In this report, a Monte Carlo code for evaluating SORS in soft tissues has been developed and compared to experimental results. The model was employed to investigate the effects of tissue and probe geometry on SORS measurements and therefore to develop the design strategies of applying SORS for breast tumor surgical margin evaluation. The model was used to predict SORS signals for different tissue geometries difficult to precisely control experimentally, such as varying normal and tumor layer sizes and the addition of a third layer. The results from the model suggest that, using source-detector separations of up to 3.75 mm, SORS can detect sub-millimeter-thick tumors under a 1 mm normal layer, and tumors at least 1 mm thick can be detected under a 2 mm normal layer.

This paper describes a handheld laser scanning confocal microscope for skin microscopy. Beam scanning is accomplished with an electromagnetic MEMS bi-axial micromirror developed for pico projector applications, providing 800 x 600 (SVGA) resolution at 56 frames per second. The design uses commercial objective lenses with an optional hemisphere front lens, operating with a range of numerical aperture from NA=0.35 to NA=1.1 and corresponding diagonal field of view ranging from 653 microm to 216 microm. Using NA=1.1 and a laser wavelength of 830 nm we measured the axial response to be 1.14 mum full width at half maximum, with a corresponding 10%-90% lateral edge response of 0.39 mum. Image examples showing both epidermal and dermal features including capillary blood flow are provided. These images represent the highest resolution and frame rate yet achieved for tissue imaging with a MEMS bi-axial scan mirror.

Infrared neural stimulation (INS) is well characterized for the peripheral nervous system; however, translation to the central nervous system (CNS) presents a new set of challenges which require us to consider different anatomy, multiple cell types, and the physiology associated with structures in the CNS. This study presents our first attempt to translate INS to in vivo stimulation of the CNS and to image the related response. The results from this study show that INS generates intrinsic optical signals of similar magnitude and shape associated with well characterized mechanical stimuli. The implications of this work could lead to neural implants which allows for single cell stimulation making it possible to design closed loop neural prosthetics.

Background and Objective: Most women with early stage breast cancer have the option of breast conserving therapy, which involves a partial mastectomy for removal of the primary tumor, usually followed by radiotherapy. The presence of tumor at or near the margin is strongly correlated with the risk of local tumor recurrence, so there is a need for a non-invasive, real-time tool to evaluate margin status. This study examined the use of autofluorescence and diffuse reflectance spectroscopy and spectral imaging to evaluate margin status intraoperatively., Materials and Methods: Spectral measurements were taken from the surface of the tissue mass immediately following removal during partial mastectomies and/or from tissues immediately after sectioning by surgical pathology. A total of 145 normal spectra were obtained from 28 patients, and 34 tumor spectra were obtained from 12 patients., Results: After correlation with histopathology, a multivariate statistical algorithm classified the spectra as normal (negative margins) or tumor (positive margins) with 85% sensitivity and 96% specificity. A separate algorithm achieved 100% classification between neo-adjuvant chemotherapy-treated tissues and non-treated tissues. Fluorescence and reflectance-based spectral images were able to demarcate a calcified lesion on the surface of a resected specimen as well., Conclusion: Fluorescence and reflectance spectroscopy could be a valuable tool for examining the superficial margin status of excised breast tumor specimens, particularly in the form of spectral imaging to examine entire margins in a single acquisition.

Raman spectroscopy has the potential to differentiate among the various stages leading to high-grade cervical cancer such as normal, squamous metaplasia, and low-grade cancer. For Raman spectroscopy to successfully differentiate among the stages, an applicable statistical method must be developed. Algorithms like linear discriminant analysis (LDA) are incapable of differentiating among three or more types of tissues. We developed a novel statistical method combining the method of maximum representation and discrimination feature (MRDF) to extract diagnostic information with sparse multinomial logistic regression (SMLR) to classify spectra based on nonlinear features for multiclass analysis of Raman spectra. We found that high-grade spectra classified correctly 95% of the time; low-grade data classified correctly 74% of the time, improving sensitivity from 92 to 98% and specificity from 81 to 96% suggesting that MRDF with SMLR is a more appropriate technique for categorizing Raman spectra. SMLR also outputs a posterior probability to evaluate the algorithm's accuracy. This combined method holds promise to diagnose subtle changes leading to cervical cancer.

Cervical cancer is the second most common malignancy among women worldwide, with over 490 000 cases diagnosed and 274 000 deaths each year. Although current screening methods have dramatically reduced cervical cancer incidence and mortality in developed countries, a "See and Treat" method would be preferred, especially in developing countries. Results from our previous work have suggested that Raman spectroscopy can be used to detect cervical precancers; however, with a classification accuracy of 88%, it was not clinically applicable. In this paper, we describe how incorporating a woman's hormonal status, particularly the point in menstrual cycle and menopausal state, into our previously developed classification algorithm improves the accuracy of our method to 94%. The results of this paper bring Raman spectroscopy one step closer to being utilized in a clinical setting to diagnose cervical dysplasia., (((c) 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).)

Background and Objectives: Nonmelanoma skin cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are the most common skin cancers, presenting nearly as many cases as all other cancers combined. The current gold-standard for clinical diagnosis of these lesions is histopathologic examination, an invasive, time-consuming procedure. There is thus considerable interest in developing a real-time, automated, noninvasive tool for nonmelanoma skin cancer diagnosis. In this study, we explored the capability of Raman microspectroscopy to provide differential diagnosis of BCC, SCC, inflamed scar tissue, and normal tissue in vivo., Study Design: Based on the results of previous in vitro studies, we developed a portable confocal Raman system with a handheld probe for clinical study. Using this portable system, we measured Raman spectra of 21 suspected nonmelanoma skin cancers in 19 patients with matched normal skin spectra. These spectra were input into nonlinear diagnostic algorithms to predict pathological designation., Results: All of the BCC (9/9), SCC (4/4), and inflamed scar tissues (8/8) were correctly predicted by the diagnostic algorithm, and 19 out of 21 normal tissues were correctly classified. This translates into a 100% (21/21) sensitivity and 91% (19/21) specificity for abnormality, with a 95% (40/42) overall classification accuracy., Conclusions: These findings reveal Raman microspectroscopy to be a viable tool for real-time diagnosis and guidance of nonmelanoma skin cancer resection.

For a given diagnostic problem, important considerations are the relative performances of the various optical biopsy techniques. A comparative evaluation of fluorescence, diffuse reflectance, combined fluorescence and diffuse reflectance, and Raman spectroscopy in discriminating different histopathologic categories of human breast tissues is reported. Optical spectra were acquired ex vivo from a total of 74 breast tissue samples belonging to 4 distinct histopathologic categories: invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), fibroadenoma (FA), and normal breast tissue. A probability-based multivariate statistical algorithm capable of direct multiclass classification was developed to analyze the diagnostic content of the spectra measured from the same set of breast tissue sites with these different techniques. The algorithm uses the theory of nonlinear maximum representation and discrimination feature for feature extraction, and the theory of sparse multinomial logistic regression for classification. The results reveal that the performance of Raman spectroscopy is superior to that of all others in classifying the breast tissues into respective histopathologic categories. The best classification accuracy was observed to be approximately 99%, 94%, 98%, and 100% for IDC, DCIS, FA, and normal breast tissues, respectively, on the basis of leave-one-sample-out cross-validation, with an overall accuracy of approximately 99%.

A common goal of most Raman spectroscopists is the acquisition of clear Raman spectra in short integration times. It is paramount, then, that the hardware used in a Raman system be optimized for throughput, signal-to-noise ratio, and resolution. To this end, we performed a systematic comparison of two Raman spectrographs: the widely used Kaiser HoloSpec and a relatively new commercial offering, the Raman Explorer by Headwall Photonics. Both strong and weak Raman scattering samples were measured using various launching conditions. When resolution-matched, the throughputs of both spectrographs were found to be roughly similar over the central range of the fingerprint region (approximately 800 to 1140 cm(-1), using 785 nm illumination), with the Raman Explorer demonstrating a slight throughput advantage outside this range. Other factors are also considered such that end users may better select the optimum spectrograph for their particular application.

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-microm intervals below the surface for each sample, down to a depth of at least 100 microm. 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.

Although skin is easily accessible to optical methodologies, a portable measurement head is necessary to allow ready spectroscopic interrogation of all anatomic locations. However, most conventional Raman microspectrometers and even dermatologic-specific Raman systems are fixed systems ill-suited to anatomic accessibility. To this end, we have developed a portable Raman microspectrometer system for future dermatologic studies. An in-house-built bench-top system was used to qualify the optical components and design. Based on this system's layout, a handheld microspectrometer was developed for future clinical application. This system produces similar operating characteristics to the bench-top prototype, and is shown to provide clear Raman spectra from skin tissue measured in vivo in clinically-feasible integration times.

Raman spectroscopy has been shown to have the potential for providing differential diagnosis in the cervix with high sensitivity and specificity in previous studies. The research presented here further evaluates the potential of near-infrared Raman spectroscopy to detect cervical dysplasia in a clinical setting. Using a portable system, Raman spectra were collected from the cervix of 79 patients using clinically feasible integration times (5 seconds on most patients). Multiple Raman measurements were taken from colposcopically normal and abnormal areas prior to the excision of tissue. Data were processed to extract Raman spectra from measured signal, which includes fluorescence and noise. The resulting spectra were correlated with the corresponding histopathologic diagnosis to determine empirical differences between different diagnostic categories. Using histology as the gold standard, logistic regression discrimination algorithms were developed to distinguish between normal ectocervix, squamous metaplasia, and high-grade dysplasia using independent training and validation sets of data. An unbiased estimate of the accuracy of the model indicates that Raman spectroscopy can distinguish between high-grade dysplasia and benign tissue with sensitivity of 89% and specificity of 81%, while colposcopy in expert hands was able to discriminate with a sensitivity and specificity of 87% and 72%.

Objective: Adult gliomas have indistinct borders. As the ratio of neoplastic cells to normal cells becomes lower, the ability to detect these cells diminishes. We describe a device designed to augment intraoperative identification of both solid tumor and infiltrating tumor margins., Methods: A novel, intraoperative, optical spectroscopic tool, using both white light reflectance and 337-nm excitation fluorescence spectroscopy, is described. Discrimination algorithms have been developed to segregate neoplastic tissues from normal glial and neuronal elements. The spectroscopy device was used to measure 5 to 10 locations during glioma resection. Beneath the tool, a biopsy sample was obtained and the pathological results were reviewed in a blinded fashion. Samples were classified as solid tumor, infiltrating tumor, or normal gray or white matter. Comparisons were made between the optical spectra and the histopathological results of sampled areas in evaluating the sensitivity and specificity of the tool for tissue discrimination., Results: Spectral data were obtained from 24 patients with glioma and from 11 patients with temporal lobe epilepsy. A sensitivity of 80% and a specificity of 89% in discriminating solid tumor from normal tissues were obtained. In addition, infiltrating tumor margins were distinguished from normal tissues with a sensitivity of 94% and a specificity of 93%., Conclusion: We have developed a handheld, optical spectroscopic device that may be used rapidly and in near real time with high sensitivity and reproducibility as an optical tissue discrimination tool in glioma surgery.

Background and Objective: Transient optical nerve stimulation is a promising new non-contact, spatially precise, artifact-free neural excitation technique useful in research and clinical settings. This study evaluates safety of this pulsed infrared laser technique by histopathologic examination of stimulated peripheral nerves., Study Design/materials and Methods: Exposed rat sciatic nerves were functionally stimulated with the pulsed Holmium:YAG laser, previously validated as an effective tool for optical stimulation. Nerves were removed immediately and up to 2 weeks after stimulation and assessed histologically for thermal damage. Laser parameters studied include upper limits for radiant exposure, repetition rate, and duration of stimulation., Results: Radiant exposures with <1% probability of thermal tissue damage (0.66-0.70 J/cm(2)) are significantly greater than radiant exposures required for reliable stimulation (0.34-0.48 J/cm(2)). The upper limit for safe laser stimulation repetition rate occurs near 5 Hz. Maximum duration for constant low repetition rate stimulation (2 Hz) is approximately 4 minutes with adequate tissue hydration., Conclusion: Results confirm that optical stimulation has the potential to become a powerful non-contact clinical and research tool for brief nerve stimulation with low risk of nerve thermal damage., ((c) 2007 Wiley-Liss, Inc.)

This paper reports the development of a probability-based spectroscopic diagnostic algorithm capable of simultaneously discriminating tumor core and tumor margins from normal human brain tissues. The algorithm uses a nonlinear method for feature extraction based on maximum representation and discrimination feature (MRDF) and a Bayesian method for classification based on sparse multinomial logistic regression (SMLR). Both the autofluorescence and the diffuse-reflectance spectra acquired in vivo from patients undergoing craniotomy or temporal lobectomy at the Vanderbilt University Medical Center were used to train and validate the algorithm. The classification accuracy was observed to be approximately 96%, 80%, and 97% for the tumor, tumor margin, and normal brain tissues, respectively, for the training data set and approximately 96%, 94%, and 100%, respectively, for the corresponding tissue types in an independent validation data set. The inherently multi-class nature of the algorithm facilitates a rapid and simultaneous classification of tissue spectra into various tissue categories without the need for a hierarchical multi-step binary classification scheme. Further, the probabilistic nature of the algorithm makes it possible to quantitatively assess the certainty of the classification and recheck the samples that are classified with higher relative uncertainty.

Objective: Adult gliomas have indistinct borders. As the ratio of neoplastic cells to normal cells becomes lower, the ability to detect these cells diminishes. We describe a device designed to augment intraoperative identification of both solid tumor and infiltrating tumor margins., Methods: A novel, intraoperative, optical spectroscopic tool, using both white light reflectance and 337-nm excitation fluorescence spectroscopy, is described. Discrimination algorithms have been developed to segregate neoplastic tissues from normal glial and neuronal elements. The spectroscopy device was used to measure 5 to 10 locations during glioma resection. Beneath the tool, a biopsy sample was obtained and the pathological results were reviewed in a blinded fashion. Samples were classified as solid tumor, infiltrating tumor, or normal gray or white matter. Comparisons were made between the optical spectra and the histopathological results of sampled areas in evaluating the sensitivity and specificity of the tool for tissue discrimination., Results: Spectral data were obtained from 24 patients with glioma and from 11 patients with temporal lobe epilepsy. A sensitivity of 80% and a specificity of 89% in discriminating solid tumor from normal tissues were obtained. In addition, infiltrating tumor margins were distinguished from normal tissues with a sensitivity of 94% and a specificity of 93%., Conclusion: We have developed a handheld, optical spectroscopic device that may be used rapidly and in near real time with high sensitivity and reproducibility as an optical tissue discrimination tool in glioma surgery.

Objective: Magnetic resonance imaging abnormalities in malignant brain tumors after irradiation may represent either recurrent tumor or radiation injury. Optical spectroscopy may represent a novel technique to identify radiation damage in brain tissues and to differentiate contrast-enhancing lesions from recurrent tumor., Methods: Fluorescence and diffuse reflectance spectra were acquired from 90 patients: 15 undergoing surgical resection for presumed recurrent tumor after radiation therapy, 15 with epilepsy and hippocampal sclerosis, and 60 with tumors who had not received irradiation. Optical spectra were acquired from 6 to 10 sites and were compared with a biopsy obtained from beneath the optical spectroscopy probe; the data then were classified by a neuropathologist blinded to the spectroscopy data. A probe for the intraoperative collection of diffuse reflectance and fluorescence spectra was used., Results: Thirteen of 15 patients (29 of 129 spectra) with previous irradiation showed a unique spectral feature characterized by a fluorescence peak centered at 500 nm (F500). All biopsy specimens showing histopathological signs of radiation injury had the F500 on their corresponding spectra (18 of 18). The F500 was identified in another 10% (11 of 111 spectra) of samples with previous irradiation but no histologically identifiable signs of radiation damage. The F500 was never seen in the normal temporal lobe of epilepsy patients with hippocampal sclerosis (0 of 105) and was seen in only 1.5% of tumor patients who did not undergo previous irradiation (6 of 433)., Conclusion: Optical spectroscopy detects radiation damage in brain tissues. The F500 spectral peak may allow accurate selection of tissues for biopsy in evaluating patients with new, contrast-enhancing lesions in the setting of previous irradiation.

Resection is not a viable treatment option for the majority of liver cancer patients. Alternatives to resection include thermotherapies such as radio-frequency ablation; however, these therapies lack adequate intraoperative feedback regarding the degree and margins of tissue thermal damage. In this proof of principle study, we test the ability of fluorescence and diffuse reflectance spectroscopy to assess local thermal damage in vivo. Spectra were acquired in vivo from healthy canine liver tissue undergoing radio-frequency ablation using a portable fiber-optic-based spectroscopic system. The major observed spectral alterations on thermal coagulation were a red shift in the fluorescence emission peak at 480 nm, a decrease in the overall fluorescence intensity, and an increase in the diffuse reflectance from 450 to 750 nm. Spectral changes were quantified and correlated to tissue histology. We found a good correlation between the proposed spectral correlates of thermal damage and histology. The results of this study suggest that fluorescence and diffuse reflectance spectroscopy show strong potential as tools to monitor liver tissue thermal damage intraoperatively., ((c) 2004 Society of Photo-Optical Instrumentation Engineers.)

There is a growing body of evidence showing that optical spectroscopy has the potential to be a useful in vivo diagnostic tool. Yet, so far there is no definitive cellular and biochemical understanding for the differences seen in the spectra from different tissue categories and disease states. In this study, we examine the use of organotypic raft cultures as an in vitro model of in vivo tissue conditions in an attempt to overcome some of the limitations of previously used methods. Organotypic raft cultures resembling normal and dysplastic epithelial cervical tissue were constructed and grown at an air-liquid interface for 2 weeks. Raman spectra of normal as well as dysplastic raft cultures were measured and compared with in vivo spectra from the corresponding tissue type. Histologic comparisons ensured that the raft cultures had similar structure and morphology to the corresponding intact tissue types. Raman spectra were also acquired from different layers of tissue. Spectral comparisons show that the Raman spectra of the raft cultures are similar to the spectra acquired from the cervix in vivo for both normal and dysplastic tissues. These results show that organotypic raft cultures are an effective and useful tool for the cellular and biochemical analysis of tissue spectroscopy.