This PDF file contains the front matter associated with SPIE Proceedings Volume 8219, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Resonant Raman spectroscopy is a technique to select and enhance the vibrational fingerprint of target molecules. Amplification of a specific Raman spectrum or specific components therein may be by several orders of magnitude. This allows to detect a molecular fingerprint even at low concentrations or in fluorescent environment without external signal enhancement. We use this approach to identify carotenoids of the antioxidant network. Quantification of the carotenoids relies on knowledge about attenuation of excitation light and Raman signal on the way through a given sample. To calculate this attenuation, the optical properties of the sample have to be known. Optoacoustics is a hybrid technique for non-invasive measurement of optical properties. We present a combined fiber sensor for optoacoustics and Raman spectroscopy allowing to probe optical properties parallel to Raman measurements. To the best of our knowledge, this is the first time that optoacoustics and vibrational spectroscopy have been combined in one sensor. It paves the way for identification and quantification of Raman active molecules in the living tissue.

Line-scan Raman microscopy (LSRM) is a versatile technique for high throughput label-free chemical mapping. The LSRM instrument achieves a hundred-fold throughput advantage over conventional point-scan Raman microscopy, by projecting a laser line onto the sample and image the Raman scattered light from the entire line using a grating spectrograph and a CCD camera. Two-dimensional chemical maps can be generated by scanning the projected line in the transverse direction. Areas of 100 x 100 μm² can be rapidly mapped with sub-micron spatial resolution and 100% fill factor. The instrument enables rapid classification of microparticles with similar shape, size and refractive index based on their chemical composition. We have achieved an equivalent imaging throughput of 100 microparticles/sec for 1 μm polystyrene beads. We have extended the technology to surface-enhanced Raman imaging, by characterizing the spatial uniformity of the SERS response of several types of nanostructured plasmonic substrates.

We report the first results of ultra-low frequency Stokes and anti-Stokes Raman spectra at 785nm showing clearly resolved frequency shifts down to 10cm^-1 from the excitation line, using commercially available ultra-narrow band notch and ASE suppression filters, and a single stage spectrometer. Near infra-red (NIR) wavelengths are of particular interest for Raman spectroscopy due to the reduced fluorescence observed for most materials. Previously reported attempts to produce ultra-low frequency Raman spectra at 785nm with volume holographic notch filters were largely unsuccessful, due to the fact that these ultra-narrow line notch filters and the wavelength of the laser must be very well matched to be effective. Otherwise, if the filters have any manufacturing errors or the laser wavelength is unstable, insufficient suppression of the Rayleigh scattered light will allow it to overwhelm the Raman signal. Recent improvements in both notch and ASE filters, wavelength-stabilized lasers, and optical system design have enabled low-frequency Raman spectra to be successfully taken at 785nm for several typical materials. Two ultra-narrow line notch filters formed as volume holographic gratings (VHGs) in glass with individually measured optical densities of 4.5 were used to block the Rayleigh scattered light from a matched VHG wavelength stabilized laser. Five discrete peaks below 100cm^-1 were simultaneously observed for sulfur in both the Stokes and anti-Stokes regions at 28, 44, 52, 62, and 83cm^-1. With no degradation in filter performance over time and extremely narrow spectral transition widths of less than 10cm^-1, this relatively simple system is able to make ultra-low frequency Stokes and anti-Stokes Raman measurements at a fraction of the size and cost of traditional triple monochromator systems.

Vibrational circular dichroism (VCD) and vibrational circular birefringence (VCB) spectra were recorded with a tunable external-cavity quantum-cascade laser (QCL). In comparison with standard thermal light sources, QCLs provide orders of magnitude more power and are therefore promising for vibrational optical activity (VOA) studies in strongly absorbing solvents and longer path length cells. The brightness of this novel light source is demonstrated with IR absorption, VCD and VCB measurements of (R)-(+)-limonene.

The leading preventable cause of death, world-wide, civilian or military, for all people between the ages of 18-45 is undetected internal hemorrhage. Autonomic compensation mechanisms mask changes such as e.g. hematocrit fluctuations that could give early warning if only they could be monitored continuously with reasonable degrees of precision and relative accuracy. Probing tissue with near infrared radiation (NIR) simultaneously produces remitted fluorescence and Raman scattering (IE) plus Rayleigh/Mie light scattering (EE) that noninvasively give chemical and physical information about the materials and objects within. We model tissue as a three-phase system: plasma and red blood cell (RBC) phases that are mobile and a static tissue phase. In vivo, any volume of tissue naturally experiences spatial and temporal fluctuations of blood plasma and RBC content. Plasma and RBC fractions may be discriminated from each other on the basis of their physical, chemical and optical properties. Thus IE and EE from NIR probing yield information about these fractions. Assuming there is no void volume in viable tissue, or that void volume is constant, changes in plasma and RBC volume fractions may be calculated from simultaneous measurements of the two observables, EE and IE. In a previously published analysis we showed the underlying phenomenology but did not provide an algorithm for calculating volume fractions from experimental data. Here we present a simple analysis that allows continuous monitoring of fluid fraction and hematocrit (Hct) changes by measuring IE and EE, and apply it to some experimental in vivo measurements.

Blood tests are an essential tool in clinical medicine with the ability diagnosis or monitor various diseases and conditions; however, the complexities of these measurements currently restrict them to a laboratory setting. P&P Optica has developed and currently produces patented high performance spectrometers and is developing a spectrometer-based system for rapid reagent-free blood analysis. An important aspect of this analysis is the need to extract the analyte specific information from the measured signal such that the analyte concentrations can be determined. To this end, advanced chemometric methods are currently being investigated and have been tested using simulated spectra. A blood plasma model was used to generate Raman, near infrared, and optical rotatory dispersion spectra with glucose as the target analyte. The potential of combined chemometric techniques, where multiple spectroscopy modalities are used in a single regression model to improve the prediction ability was investigated using unfold partial least squares and multiblock partial least squares. Results show improvement in the predictions of glucose levels using the combined methods and demonstrate potential for multiblock chemometrics in spectroscopic blood analysis.

Visualization of cells and subcellular organelles are currently carried out using available microscopy methods such as cryoelectron microscopy, and fluorescence microscopy. These methods require external labeling using fluorescent dyes and extensive sample preparations to access the subcellular structures. However, Raman micro-spectroscopy provides a non-invasive, label-free method for imaging the cells with chemical specificity at sub-micrometer spatial resolutions. The scope of this paper is to image the biochemical/molecular distributions in cells associated with cancerous changes. Raman map data sets were acquired from the human cervical carcinoma cell lines (HeLa) after fixation under 785 nm excitation wavelength. The individual spectrum was recorded by raster-scanning the laser beam over the sample with 1μm step size and 10s exposure time. Images revealing nucleic acids, lipids and proteins (phenylalanine, amide I) were reconstructed using univariate methods. In near future, the small pixel to pixel variations will also be imaged using different multivariate methods (PCA, clustering (HCA, K-means, FCM)) to determine the main cellular constitutions. The hyper-spectral image of cell was reconstructed utilizing the spectral contrast at different pixels of the cell (due to the variation in the biochemical distribution) without using fluorescent dyes. Normal cervical squamous cells will also be imaged in order to differentiate normal and cancer cells of cervix using the biochemical changes in different grades of cancer. Based on the information obtained from the pseudo-color maps, constructed from the hyper-spectral cubes, the primary cellular constituents of normal and cervical cancer cells were identified.

Multivariate classifiers (such as Linear Discriminant Analysis, Support Vector Machines etc) are known to be useful tools for making diagnostic decisions based on spectroscopic data. However, robust techniques for assessing their performance (e.g. by sensitivity and specificity) are vital if the application of these methods is to be successful in the clinic. In this work the application of repeated cross-validation for estimating confidence intervals for sensitivity and specificity of multivariate classifiers is presented. Furthermore, permutation testing is presented as a suitable technique for estimating the probability of obtaining the observed sensitivity and specificity by chance. Both approaches are demonstrated through their application to a Raman spectroscopic model of gastrointestinal cancer.

Nonenzymatic glycation and oxidation of ubiquitous proteins in vivo leads to irreversible formation of advanced glycation end products (AGEs). Due to their relatively long half life and low clearance rate AGEs tend to accumulate within static tissues and the circulatory system. Spectra obtained using 830 nm near-infrared (NIR) excitation suggest that the so-called "autofluorescence" from all tissues has a finite number of sources but the fact that senior and diabetic subjects produce more than other members of the general population suggests that a significant portion of the total autofluorescence from all sources originates from AGEs. Using pentosidine generated in a reaction mixture as described by Monnier as representative, an in vitro study unveiled very similar fluorescence and photobleaching pattern as observed for autofluorescence in vivo. A series of oxygen, air and argon purging experiments on the pentosidine-generating reaction mixture suggests that pentosidine is a singlet oxygen sensitizer and secondary reactions between the pentosidine itself and/or other fluorophores and the photosensitized singlet oxygen explain the observed photobleaching. Ab initio Gaussian calculations on pentosidine reveal the existence of low-lying triplet excited states required for the sensitization of ground state oxygen. A commercially available product known as singlet oxygen sensor green (SOSG) that specifically serves as a singlet oxygen detection reagent confirms the generation of singlet oxygen from NIR irradiated pentosidine trimixture. This study provides one definite chemical mechanism for understanding in vivo human skin autofluorescence and photobleaching.

Raman spectroscopy is a non-invasive technique offering great potential in the biomedical field for label-free discrimination between normal and tumor cells based on their biochemical composition. First, this contribution describes Raman spectra of lymphocytes after drying, in laser tweezers, and trapped in a microfluidic environment. Second, spectral differences between lymphocytes and acute myeloid leukemia cells (OCI-AML3) are compared for these three experimental conditions. Significant similarities of difference spectra are consistent with the biological relevance of the spectral features. Third, modulated wavelength Raman spectroscopy has been applied to this model system to demonstrate background suppression. Here, the laser excitation wavelength of 785 nm was modulated with a frequency of 40 mHz by 0.6 nm. 40 spectra were accumulated with an exposure time of 5 seconds each. These data were subjected to principal component analysis to calculate modulated Raman signatures. The loading of the principal component shows characteristics of first derivatives with derivative like band shapes. The derivative of this loading corresponds to a pseudo-second derivative spectrum and enables to determine band positions.

Using integrated Raman and angular scattering microscopy (IRAM), we follow the response of EMT6 cancer cells to photodynamic therapy (PDT) treatment. The study combines two non-labelling light scattering techniques to extract chemical information and organelle sizes from single cells. Each cell is measured repeatedly over several hours to follow changes in these parameters as the cell responds to the PDT treatment. An automated algorithm identifies which parameters are changing in time. Size parameters extracted from angular scattering measurements show a decrease in the size of 1-micron-diameter scatterers in treated cells. Treated cells also exhibit trends in several Raman peaks, denoting changes in chemical concentrations of proteins, nucleic acids, and lipids. Each of these parameters - acquired from both measurement modalities - can be monitored on a cell-by-cell basis. The ability to track these chemical and structural changes over time allows access to greater knowledge of biological processes.

We show the application of near-infrared Raman Spectroscopy to in-vitro monitoring of the viability of tissue constructs (EVPOMEs). During their two week production period EVPOME may encounter thermal, chemical or biochemical stresses that could cause development to cease, rendering the affected constructs useless. We discuss the development of a Raman spectroscopic technique to study EVPOMEs noninvasively, with the ultimate goal of applying it in-vivo. We identify Raman spectroscopic failure indicators for EVPOMEs, which are stressed by temperature, and discuss the implications of varying calcium concentration and pre-treatment of the human keratinocytes with Rapamycin. In particular, Raman spectra show correlation of the peak height ratios of CH₂ deformation to phenylalanine ring breathing, providing a Raman metric to distinguish between viable and nonviable constructs. We also show the results of singular value decomposition analysis, demonstrating the applicability of Raman spectroscopic technique to both distinguish between stressed and non-stressed EVPOME constructs, as well as between EVPOMEs and bare AlloDerm® substrates, on which the oral keratinocytes have been cultured. We also discuss complications arising from non-uniform thickness of the AlloDerm® substrate and the cultured constructs, as well as sampling protocols used to detect local stress and other problems that may be encountered in the constructs.

Oral squamous cell carcinoma is sixth among the major malignancies worldwide. Tobacco habits are known as major causative factor in tumor carcinogenesis in oral cancer. Optical spectroscopy methods, including Raman, are being actively pursued as alternative/adjunct for cancer diagnosis. Earlier studies have demonstrated the feasibility of classifying normal, premalignant and malignant oral ex-vivo tissues. In the present study we have recorded in vivo spectra from contralateral normal and diseased sites of 50 subjects with pathologically confirmed lesions of buccal mucosa using fiber-optic-probe-coupled HE-785 Raman spectrometer. Spectra were recorded on similar points as per teeth positions with an average acquisition time of 8 seconds. A total of 215 and 225 spectra from normal and tumor sites, respectively, were recorded. Finger print region (1200-1800 cm^-1) was utilized for classification using LDA. Standard-model was developed using 125 normal and 139 tumor spectra from 27 subjects. Two separate clusters with an efficiency of ~95% were obtained. Cross-validation with leave-one-out yielded ~90% efficiency. Remaining 90 normal and 86 tumor spectra were used as test data and predication efficiency of model was evaluated. Findings of the study indicate that Raman spectroscopic methods in combination with appropriate multivariate tool can be used for objective, noninvasive and rapid diagnosis.

Raman spectroscopy is an inelastic scattering technique capable of probing the biochemical changes associated with neoplastic progression in oesophageal tissue. Custom-built fibre-optic Raman probes could potentially provide opportunities for in vivo endoscopic diagnosis of pre-cancerous oesophageal lesions and targeted early therapy. However, prior to commencing a clinical trial convincing ex vivo work must demonstrate multi-operator, multi-centre and multi-system reliability. We report spectral consistency between two operators who independently evaluated two optically identical probes ex vivo. In addition, we demonstrate compatibility with high-definition white light endoscopes and narrow band imaging systems highlighting the potential for future endoscopic multi-modality imaging in the oesophagus.

Raman spectroscopy is a vibrational analytic technique sensitive to the changes in biomolecular composition and conformations occurring in tissue. With our most recent development of near-infrared (NIR) Raman endoscopy integrated with diagnostic algorithms, in vivo real-time Raman diagnostics has been realized under multimodal wide-field imaging (i.e., white- light reflectance (WLR), narrow-band imaging (NBI), autofluorescence imaging (AFI)) modalities. A selection of 177 patients who previously underwent Raman endoscopy (n=2510 spectra) was used to render two robust models based on partial least squares - discriminant analysis (PLS-DA) for esophageal and gastric cancer diagnosis. The Raman endoscopy technique was validated prospectively on 4 new gastric and esophageal patients for in vivo tissue diagnosis. The Raman endoscopic technique could identify esophageal cancer in vivo with a sensitivity of 88.9% (8/9) and specificity of 100.0% (11/11) and gastric cancers with a sensitivity of 77.8% (14/18) and specificity of 100.0% (13/13). This study realizes for the first time the image-guided Raman endoscopy for real-time in vivo diagnosis of malignancies in the esophagus and gastric at the biomolecular level.

Although glucocorticoids are among the most frequently prescribed anti-inflammatory agents used in the treatment of rheumatoid arthritis, extended exposure to this steroid hormone is the leading cause of iatrogenic osteoporosis. Recently, Raman spectroscopy has been utilized to exploit biochemical differences between osteoporotic and normal bones in order to predict fracture risk. In this presentation, we report the results of ongoing research in our laboratory towards the clinical translation of this technique. We will discuss strategies for the transcutaneous acquisition of spectra from the tibiae of mice that are of sufficient quality to generate accurate predictions of fracture risk.

Histopathology is the gold standard for disease diagnosis; however it is subject to a number of limitations. Fourier Transform infrared (FT-IR) spectroscopic imaging can be used to derive chemical images from tissues based on their inherent molecular composition, thereby eliminating the use of dyes and stains. FT-IR imaging represents a novel, emerging approach that can allow for accurate cell type identification which is competitive with conventional histopathological approaches and may alleviate a number of the limitations associated with current techniques. Traditionally, this approach has involved in a loss of image detail due to the sub-optimal and, compared to optical microscopy, coarse pixel size in instruments. Recent advances in high-resolution FT-IR imaging have allowed for the identification and chemical characterization of cell types and tissue structures which were previously not discernible. Here we report on the visualization of several histologic details using high-resolution IR imaging that may be critical for tissue histology and disease diagnosis.

Diabetes mellitus is a disorder of glucose metabolism and it is one of the most challenging diseases, both from a medical and economic perspective. People with diabetes can benefit from a frequent or even continuous monitoring of their blood glucose concentrations. The approach presented here takes advantage of the observational nature of biomedical vibrational spectroscopy in contrast to chemical reactions which consume glucose. The particular technique employed here is based on the high sensitivity of mid-infrared transmission spectroscopy where strong vibrational bands of glucose can be monitored at wavelengths around 10 μm. The strong absorption of water in this spectral region was mitigated by the use of quantum cascade lasers and very short interaction path lengths below 50 μm. Various sensor concepts have been explored. In one of the concepts, the interaction of mid-infrared radiation with glucose is established within a miniature measurement cavity, formed by a gap between two silver halide fibers. In recent experiments, an additional quantum cascade laser was used for reference purposes. The long-term drift could significantly be reduced for time intervals > 1000 s, e. g., by more than 60% for a 3 hour interval. This extension for the compensation of long-term drifts of the measurement system in vitro is an important contribution towards the applicability in vivo.

Paracoccidioides brasiliensis (P. brasiliensis) is a thermal dimorphic fungus and causal agent of paracoccidioidomycosis. Epidemiological data shows that it is mainly concentrated in Central and South America countries, with most registered cases in Colombia, Brazil, and Venezuela. The histopathological similarity with others fungal infection makes the diagnosis of P. brasiliensis more complicated. Therefore, the aim of this work was to find a positive and negative test for P. brasiliensis using gold nanoprobes as a new tool for P. brasiliensis detection. Gold nanoparticles were synthesized by reduction of gold chloride with sodium citrate. The results of this procedure is a wine-red solution with a maximum absorption in the range of ~520-530nm. A specific P. brasiliensis sequence of oligonucleotide was bonded to the nanoparticles, which maintained the wine-red color. The color changes from red to blue for negative diagnostic and is unchanged for a positive test. The H-bond interaction of DNA with the complementary DNA keeps strands together and forms double helical structure, maintaining the colloid stability. However, for non-complimentary DNA sequence the nanoprobes merge into a cluster, changing the light absorption.

Rheumatoid arthritis is a systemic inflammatory disease of unknown causes and a new methods to identify it in early stages are needed. The main purpose of this work is the biochemical differentiation of sera between normal and RA patients, through the establishment of a statistical method that can be appropriately used for serological analysis. The human sera from 39 healthy donors and 39 rheumatics donors were collected and analyzed by Fourier Transform Infrared Spectroscopy. The results show significant spectral variations with p<0.05 in regions corresponding to protein, lipids and immunoglobulins. The technique of latex particles, coated with human IgG and monoclonal anti-CRP by indirect agglutination known as FR and CRP, was performed to confirm possible false-negative results within the groups, facilitating the statistical interpretation and validation of the technique.

Paracoccidioides brasiliensis the etiological agent of paracoccidioidomycosis, is a dimorphic fungus existing as mycelia in the environment (or at 25 °C in vitro) and as yeast cells in the human host (or at 37°C in vitro). The most prominent difference between both forms is probably the cell wall polysaccharide, being 1,3-β-glucan usually found in mycelia and 1,3-α-glucan found in yeasts, but a plethora of other differences have already been described. In this work, we performed a Fourier Transform Infrared Spectroscopy analysis to compare the yeast and mycelia forms of P. brasiliensis and found additional biochemical differences. The analysis of the spectra showed that differences were distributed in chemical bonds of proteins, lipids and carbohydrates.

Rapid microbiological identification and characterization are very important in dentistry and medicine. In addition to dental diseases, pathogens are directly linked to cases of endocarditis, premature delivery, low birth weight, and loss of organ transplants. Fourier Transform Infrared Spectroscopy (FTIR) was used to analyze oral pathogens Aggregatibacter actinomycetemcomitans ATCC 29523, Aggregatibacter actinomycetemcomitans-JP2, and Aggregatibacter actinomycetemcomitans which was clinically isolated from the human blood-CI. Significant spectra differences were found among each organism allowing the identification and characterization of each bacterial species. Vibrational modes in the regions of 3500-2800 cm^-1, the 1484-1420 cm^-1, and 1000-750 cm^-1 were used in this differentiation. The identification and classification of each strain were performed by cluster analysis achieving 100% separation of strains. This study demonstrated that FTIR can be used to decrease the identification time, compared to the traditional methods, of fastidious buccal microorganisms associated with the etiology of the manifestation of periodontitis.

Although there are many articles focused on in vivo or ex vivo Raman analysis for cancer diagnosis, to the best of our knowledge its potential to predict the aggressiveness of tumor has not been fully explored yet. In this work Raman spectra in the finger print region of ex vivo breast tissues of both healthy mice (normal) and mice with induced mammary gland tumors (abnormal) were measured and associated to matrix metalloproteinase-19 (MMP-19) immunohistochemical exam. It was possible to verify that normal breast, benign lesions, and adenocarcinomas spectra, including the subtypes (cribriform, papillary and solid) could have their aggressiveness diagnosed by vibrational Raman bands. By using MMP- 19 exam it was possible to classify the samples by malignant graduation in accordance to the classification results of Principal Component Analysis (PCA). The spectra NM /MH were classified correctly in 100% of cases; CA/CPA group had 60 % of spectra correctly classified and for PA/AS 54% of the spectra were correctly classified.