Different cluster image reassembling methodologies have been used to generate infrared maps from FT-IR microspectra of human prostate tissue sections. Spectra were collected in transmission mode with high spatial resolution by the use of a HgCdTe focal plane array detector imaging system. While univariate imaging techniques such as chemical mapping often give unsatisfactory classification results, unsupervised multivariate data analysis techniques such as agglomerative hierarchical clustering, fuzzy C-means, or k-means clustering confirmed standard histopathological techniques and turned out to be helpful to identify and to discriminate tissues structures. The use of any of the clustering algorithms dramatically increased the information content of the IR images, as compared to chemical mapping. Among the cluster imaging methods, agglomerative hierarchical clustering (Ward's algorithm) turned out to be the best method in terms of tissue structure differentiation.

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.

Syrian hamster nervous tissue was investigated by FTIR microspectroscopy with conventional and synchrotron infrared light sources. Various tissue structures from the cerebellum and medulla oblongata of scrapie-infected and control hamsters were investigated at a spatial resolution of 50 μm. Single neurons in dorsal root ganglia of scrapie-infected hamsters were analyzed by raster scan mapping at 6 μm spatial resolution. These measurements enabled us to (i) scrutinize structural differences between infected and non-infected tissue and (ii) analyze for the first time the distribution of different protein structures in situ within single nerve cells. Single nerve cells exhibited areas of increased β-sheet content, which co-localized consistently with accumulations of the pathological prion protein (PrP^Sc). Spectral data were also obtained from purified, partly proteinase K digested PrP^Sc isolated from scrapie-infected nervous tissue of hamsters to elucidate similarities/dissimilarities between prion structure in situ and ex vivo. A further comparison is drawn to the recombinant Syrian hamster prion protein SHaPrP^90-232, whose in vitro transition from the predominantly a-helical isoform to β-sheet rich oligomeric structures was also investigated by FTIR spectroscopy.

We tested the hypothesis that FT-IR spectrometry was useful for determining oxidative stress damage on erythrocytes. Endurance-trained subjects performed a standardized endurance-training session at 75% of maximal oxygen consumption each week over 19 consecutive weeks. Capillary blood samples were taken before and after test-sessions and plasma and erythrocytes were separately analyzed using Fourier-transform infrared spectrometry. Exercise-induced change in plasma concentrations and erythrocyte IR absorptivities (vC-H_n of fatty acyl moieties, vC=O and δN-H of proteins, vP=O of phospholipids, vCOO^- of amino-acids, and vC-O of lactate) were monitored and compared to training level. First training weeks induced normalization of plasma concentration changes during exercise (unchanged for glucose, moderately increased for lactate, high increases for triglycerides, glycerol, and fatty acids) while erythrocyte phospholipids alteration remained elevated (P < 0.05). Further, training reduced the exercise-induced erythrocyte lactate content increase (vC-O; P < 0.05) and phospholipids alteration (vC-H_n and vP=O; P < 0.05) during exercise. These changes paralleled the lowering of exercise-induced hemoconcentration (P < 0.05) and plasma lactate concentration increase during exercise (P < 0.05). These correlated changes between plasma and erythrocyte parameters suggest that hemoconcentration and lactate acidosis (plasmatic and intracellular) are important factors contributing to reduce erythrocyte susceptibility to oxidative stress during chronic endurance training.

Fiber Evanescent Wave Spectroscopy (FEWS) is a very useful method for non-invasive and non-destructive biomedical diagnosis. We have developed a FEWS system that makes use of a Fourier Transform Infrared (FTIR) spectrometer and IR transmitting AgBr_xCl_1-x fibers. The FTIR-FEWS system is compact and easy to use, and it is ideal for the study of the spectroscopy of the skin in the mid-IR. The evanescent wave penetration depth in the mid-IR is comparable with the thickness of the stratum corneum, and therefore the vibrational spectra of lipids, proteins and water can be easily analyzed. We have used FTIR-FEWS for a clinical study of the skin of 60 patients, who had some suspicious skin lesions. Preliminary measurements were carried out both on the lesion and on neighboring healthy areas of the skin, showing some differences in the IR absorption. More data is needed in order to determine the possibility of diagnosis of skin cancer and its type from mid-IR spectral data.

Fiberoptic Evanescent Wave Spectroscopy (FEWS) has been used for measurements of the absorption of very small (0.1mg) fragments of urinary calculi in the mid-IR. Such measurements were used for the determination of the chemical composition of each fragment. When large urinary stones are fragmented, it is possible to use this method for determining the chemical composition of the inner part and the outer part of the stone. We examined 40 urinary calculi and found that in 1/3 of them the inner part and the outer parts are identical. In 2/3 the inner part is different than the outer part. This change is not revealed by standard chemical methods that provide an average chemical composition. The novel FEWS method would be useful for the analysis of urinary calculi.

Design and functionality of a reagent-free Raman-based fiber optic sensor are described in the context of glucose monitoring. Theoretical calculations of sensor performance are shown using raytrace software. An optimized sensor design with an outer diameter of 1.5 mm was carried out. First experiments in aqueous glucose solutions showed a root mean square error of prediction of 10.5 mg/dl in an independent validation.

Raman and FTIR microprobe spectroscopy have been used to characterize the atherosclerotic process in Apo E and wild type mice. The Apo E null mouse is being studied in parallel with a healthy strain as a model of the human atherosclerotic disease. Preliminary Raman microprobe spectra have been recorded from the lumen of the aorta vessels from a normal black mouse (C57BL/6J) and the apo E null mouse fed on a normal chow diet. Spectra were also recorded from another normal mouse fed breeder chow containing a much higher content of fats. In the Raman spectra the fat cells exhibited spectra typical of esterified triglycerides while the wall tissue had spectra dominated by Amide I and III modes and the phenylalanine stretch at 1003 cm^-1 of protein. The FTIR spectra showed the typical Amide I and II bands of protein and the strong >C=O stretch of the triglycerides. In addition, there were morphologically distinct regions of the specimens indicating a surprising form of calcification in one very old mouse (wild type), and free fatty acid inclusions in the knock out mouse. The observation of these chemistries provide new information for elucidation of the molecular mechanisms of the development of atherosclerosis.

Osteogenesis imperfecta (OI) is genetic defect in which the genes that code for the α1(I) or α2(I) chains of type I collagen are defective. The defects often result in substitution of a bulky amino acid for glycine, causing formation of collagen that can not form the normal triple helix. Depending on the details of the defects, the outcomes range from controllable to lethal. This study focuses on OI type IV, a more common and moderately severe form of the disease. People with the disease have a substantial increase in the risk and rate of fracture. We examine the spectroscopic consequences of these defects, using a mouse model (BRTL) that mimics OI type IV. We compare Raman images from tibial cortical tissue of wild-type mice and BRTL mice with single copy of mutation and show that both mineral to matrix ratios and collagen inter-fibril cross-links are different in wild-type and mutant mice.

The incidence of both prostate and bladder cancer is high; prostate cancer being the most frequently diagnosed non-cutaneous cancer affecting Western men. At present the gold standard for diagnosis of pathologies within the bladder and the prostate gland is by means of histological examination of biopsies. This is a subjective means of examining tissue and has an element of both inter and intra-observer variability. A large number of specimens have been collected and analysed using both a NIR-Raman spectrometer and histopathology with H&E staining. Multivariate spectral prediction models have been constructed and tested. An evaluation of misclassification cost models and the use of cancer staging data to train the models has been made.

Raman Spectroscopy is an optical diagnostic technique applied in this study to classify axillary lymph nodes from breast cancer patients as positive or negative for metastases. The mapping technique in this study is 81% sensitive and 97% specific for the correct classification of positive lymph nodes. Raman spectral images of lymph node sections are constructed to facilitate interpretation of tissue features.

FT-Raman Spectroscopy (FTRS) has being applied as a new diagnostic tool for the early detection of oral cancer, showing spectral sensitivity to changes in the molecular composition and morphology associated with malignant tissues. A carcinogen (7, 12-dimethybenz[a]anthracene-DMBA) was applied daily in the buccal pouch of 23 hamster and after 14 days fragments of chemically-induced and normal tissue were analyzed by FTRS. A 1064nm, 100mW Nd:YAG laser was used as excitation source. A total of 123 spectra were obtained (three in each site, left and right pouch). They were divided into normal and malignant tissue groups according to the anatomopathologist and were analyzed statistically based on Principal Components Analysis (PCA). The spectra region of 1263-1337 cm^-1 showed significant difference between the normal and squamous cell carcinomas samples. The region of 1555-1560 cm^-1 also has showed an increase of intensity in the malignant samples. An algorithm based on Principal Components Analysis could separate the samples in two groups, obtaining 86% of sensitivity and 93% of specificity, for the training group, while the prospective group had 85% of sensitivity and 88 % of specificity. Results suggest that the FT-Raman spectroscopy can be useful for detection of malign oral lesion in an animal model.

The identification and analysis of disease-specific signatures in mid-infrared spectra of serum forms the basis of a method called “Diagnostic Pattern Recognition (DPR)”. A conceivable usage of this method in clinical diagnostics requires that the method be applied in a convenient and robust manner. Thus, automation, room-temperature operation and reproducibility the prerequisite improvements toward routine application. We have investigated the performance two identical, semi-automated DPR systems. In contrast to previous measurements, which required MCT detectors, the use of a DLaTGS detector allowed the systems to be operated without the requirement of liquid nitrogen cooling. A series of measurements showed that automated pipetting improves the reproducibility significantly as compared to manual pipetting. For automated pipetting, the within-day variations are of minor importance. However, day-to-day variations may decrease the reproducibility in some spectral regions by more than a factor of two. Slight dependence of the reproducibility on the protein content of the serum samples has been observed.

The mid-infrared wavelength region contains characteristic peaks of several of the biochemical constituents of tissue. Recently, it has been shown that measurements of mammalian cell suspensions can provide estimates of biochemical composition and consequently information on the growth stage. This information may be used to identify cancerous tissue in-vivo. To facilitate the development of an in-vivo diagnostic technique, we have performed simulations of photon propagation and light collection in epithelial tissue, given a specific optical probe geometry.

A time-resolved diffuse reflectance from a semi-infinite homogeneous medium is compressed along the time axis and multiplied by appropriate factors. According to the photon diffusion equation, a gradient of the attenuation difference between the compressed reflectance and that measured with a smaller source-detector distance is proportional to the absorption coefficient. Using this property, a simple algorithm using spatially and time-resolved reflectance to measure the absorption coefficient of a homogeneous medium is proposed as an alternative to the procedure of fitting to the photon diffusion equation. In the case of a two-layered medium, the absorption coefficient of each layer can be estimated also using this simple algorithm if approximate values of the depth of the upper layer and the scattering coefficients of the two layers are known beforehand. For validation experiment, the time-resolved reflectance from a polyacetal block was measured at various source-detector distances and the estimated absorption coefficient of the block was compared to that obtained using the conventional method. As the result of in vivo experiment, the absorption coefficient of the lower layer of a human head was found to be larger than that of the upper layer under the assumption that the human head consisted of two layers.

Vibrationally-sensitive spectroscopic techniques are becoming important clinical tools for real-time, in vivo diagnostics. The molecular information made available with these techniques can provide early diagnostic signs of disease, often before morphological changes occur. We model and experimentally demonstrate a new technique for measuring optical spectroscopy signals using interferometric ranging. This new technique, nonlinear interferometric vibrational imaging (NIVI), uses principles from coherent anti-Stokes Raman scattering (CARS) spectroscopy and optical coherence tomography (OCT) to achieve cross-sectional imaging of the distribution of specific molecular species within a sample. Two CARS signals are generated, one from a known reference molecular species and a second from the unknown molecules in a sample. These coherent signals are interfered with each other using an interferometer setup. The intensity envelope of the interference signal provides a measure of the concentration of selected bonds present in the sample focal volume. The interference fringes themselves can provide phase information that will allow for the exact reconstruction of the vibrational characteristics of the molecules in the sample focal volume. Theoretical background to CARS interferometry is presented, the experimental laser systems are described, and a depth-resolved scan line of a benzene filled cuvette is demonstrated. The experimental results show close resemblance to the theoretical models. The advantages of NIVI over existing vibrational imaging systems and its clinical implications are discussed.

We propose a methodology for enhancing the diffraction limited spatial resolution attained in Raman and Fourier transform infrared microspectroscopic imaging techniques. Near-field scanning optical microscopy (SNOM) and spectroscopy employ apertureless and aperture approaches to provide ultra-high spatially resolved images at the nanometer level. In contrast, we employ conventional spectral acquisition schemes modified by spatial oversampling with the subsequent application of deconvolution techniques. As an example, this methodology is applied to flat samples using point illumination. Simulated data, assuming idealized sample concentration profiles, are presented together with experimental Raman microspectroscopic data from chemically and morphologically well-defined test samples. Intensity profiles determined using conventional mapping and imaging techniques are compared to those obtained by the probe/deconvolution methodology.

We show that Raman spectroscopy with visible lasers, even in the deep blue is possible with time-gated Raman spectroscopy. A 4 picosec time gate allows efficient fluorescence rejection, up to 1000X, and provides almost background-free Raman spectra with low incident laser power. The technology enables spectroscopy with better than 10X higher scattering efficiency than is possible with the NIR (785 nm and 830 nm) lasers that are conventionally used. Raman photon migration is shown to allow depth penetration. We show for the first time that Kerr-gated Raman spectra of bone tissue with blue laser excitation enables both fluorescence rejection and depth penetration.

The 2-2.5μm region of the electromagnetic spectrum is of particular importance for the non-invasive monitoring of blood glucose using absorption spectroscopy, since it can provide the strongest signature as compared to other water transmission windows. Currently available spectroscopy systems for this application require high-gain and low-noise detectors in order to achieve sufficient signal-to-noise ratio measurements. In this context, we are investigating the integration of micro-optics along with InGaAsSb/AlGaAsSb avalanche photodetectors in order to demonstrate high-fill factor, high quantum efficiency and eventually the ability to evaluate the blood glucose concentration with high accuracy. Also, using the bandgap engineering options afforded by the quaternary antimonide structures, the spectral response of the detector can be tuned over this wavelength range. In this paper, we present the design, fabrication and integration of the multi-chip modules, the constituent technologies required to realize them and experimental results from their characterization.

Optical spectroscopy has been extensively studied as a potential in vivo diagnostic tool to provide information about the chemical and morphologic structure of tissue. Raman Spectroscpy is an inelastic scattering process that can provide a wealth of spectral features that can be related to the specific molecular structure of the sample. This article reports results of an in vitro study of the FT-Raman human breast tissue spectra. An Nd:YAG laser at 1064nm was used as the excitation source in the FT-Raman Spectrometer. The neoplastic human breast samples, both Fibroadenoma and ICD, were obtained during therapeutical routine medical procedures required by the primary disease, and the non-diseased human tissue was obtained in plastic surgery. No sample preparation was needed for the FT-Raman spectra collection. The FT-Raman spectra were recorded from normal, benign (Fibroadenomas) and malignant (IDC-Intraductal Carcinoma) samples, adding up 51 different areas. The main spectral differences of a typical FT-Raman spectra of a Normal (Non-diseased), Fibroadenoma, and Infiltrating Ductal Carcinoma (IDC) breast tissue at the interval of 600 to 1800cm^-1, which may differentiate diagnostically the sample, were found in the bands of 1230 to 1295cm^-1, 1440 to 1460 cm^-1 and 1650 to 1680 cm^-1, assigned to the vibrational bands of the carbohydrate-amide III, proteins and lipids, and carbohydrate-amide I, respectively.

FT-Raman spectroscopy is a modern analytical tool and it is believed that its use for skin cancer diagnosis will lead to several advantages for patients, e.g., faster results and a minimization of invasivity. This article reports results of an ex Vivo study of the FT-Raman spectra regarding differentiation between non-diseased and malignant human skin lesions, Basal Cell Carcinoma (BCC). A Nd: YAG laser at 1064nm was used as the excitation source in the FT-Raman, RFS 100/S Spectrometer, Bruker. Thirty-nine sets of human skin samples, 18 histopathologically diagnosed as non-diseased, and 21 as BCC, were obtained during routine therapeutic procedures required by the primary disease. No sample preparation was needed to promote the FT-Raman spectra collection. The main spectral features, which may differentiate the sample, were found in the shift region of Amide I (1640 to 1680 cm^-1), Amide III (1220 to 1330cm^-1), proteins and lipids (1400 to 1500 cm^-1), amino acids (939 to 940 cm^-1) and deoxyribonucleic acid (1600 to 1620cm^-1). Principal Components Analysis (PCA) was applied to FT-Raman spectra of Basal Cell Carcinoma. Analysis was performed on mean-normalized and mean-centered data of the non-diseased skin and BCC spectra. The dynamic loading of PCA was expanded into 2D contour by calculating a variance-covariance matrix. PCA was used to verify the statistical differences in the sample. This technique applied over all samples identified tissue type within 83% of sensitivity and 100% specificity. The PCA technique proved efficient for analysis in skin tissue ex vivo, results were significant and coherent.

FT- Raman Spectroscopy (FT-Raman) could allow identification and evaluation of human atherosclerotic lesions. A Raman spectrum can provide biochemical information of arteries which can help identifying the disease status and evolution. In this study, it is shown the results of FT-Raman for identification of human carotid arteries in vitro. Fragments of human carotid arteries were analyzed using a FT-Raman spectrometer with a Nd:YAG laser at 1064nm operating at an excitation power of 300mW. Spectra were obtained with 250 scans and spectral resolution of 4 cm^-1. Each collection time was approximately 8 min. A total of 75 carotid fragments were spectroscopically scanned and FT-Raman results were compared with histopathology. Principal Components Analysis (PCA) was used to model an algorithm for tissue classification into three categories: normal, atherosclerotic plaque without calcification and atherosclerotic plaque with calcification. Non-atherosclerotic (normal) artery, atherosclerotic plaque and calcified plaque exhibit different spectral signatures related to biochemicals presented in each tissue type, such as, bands of collagen and elastin (proteins), cholesterol and its esters and calcium hydroxyapatite and carbonate apatite respectively. Results show that there is 96% match between classifications based on PCA algorithm and histopathology. The diagnostic applied over all 75 samples had sensitivity and specificity of about 89% and 100%, respectively, for atherosclerotic plaque and 100% and 98% for calcified plaque.

As a part of non-invasive and unaware measurement of physiological signal in the house of live-alone person, Raman spectroscopy was applied for urine component analysis in the toilet set. 785nm, 250-300mW output solid state diode laser and 2048 element linear silicon TE cooled CCD array were incorporated for this system. Several tests were performed for setting up Raman spectroscopy in non-constrained situation: toilet set in the house. The effect of dark current, integration time, warming up time of laser, property of probe and interference of water in the toilet were tested and controlled for appropriate measurement in this environment. The spectra were obtained immediately when the subject uses the toilet set, and they can be transmitted to the server though Bluetooth. Those spectra were pre-processed for removing or correcting the effect of undesired light scattering, sample path-length difference and baseline-effect. The preprocessed data were enhanced for more exact result of multivariate analysis. The training data was prepared for predicting unknown component and its concentration by using multivariate methods. Several kinds of multivariate methods: PCA, PCR, PLS were performed to validate what is the fittest method in this environment. Through quantitative and qualitative analysis of Raman spectroscopy’s spectra obtained in the house's toilet set, we could know the component and its concentration of urine which can be index of disease.

As part of an ongoing larger study of the molecular and supramolecular foundations of bone tissue biomechanics, we report thermal perturbations to bone mineral and related model compounds. The response of bone tissue to external mechanical and thermal loading under a variety of conditions is used to elucidate the response to physiologically relevant loads. Here NMR spectroscopy is used in conjunction with Raman spectroscopy to elucidate the mineral structure of the bone and track changes in the lattice due to temperature variation. Changes in the bone lattice are studied by examining the Raman spectral band widths and positions of the phosphate and carbonate bands. Expansion of the lattice leads to increased band widths as local ion motion is facilitated. Larger effects are found in undeproteinated bone powder than in deproteinated bone mineral powder. 1H MAS NMR is used to track the water content of deproteinated bone as a function of temperature. The differing effects observed in undeproteinated bone powder and deproteinated bone mineral powder suggest that mineral crystallite expansion may involve mechanical constraint by the bone matrix. 13C MAS NMR spectroscopy revealed a loss of carbonate in deproteinated bone mineral when heated to 225 C. This is a significantly lower temperature than previously reported for removal of carbonate from synthetic apatite material. The properties of bone mineral influenced by even small perturbations such as temperature elevation or reduction depend on the presence of matrix. It is reasonable to assume that bone tissue response to other external loads, including compression or bending under normal physiological conditions also depend on the interaction of mineral and matrix.

Raman spectroscopy is used as a probe of ultrastructural (molecular) changes in both the mineral and matrix (protein and glycoprotein, predominantly type I collagen) components of murine cortical bone as it responds to loading in the elastic regime. At the ultrastructural level, crystal structure and protein secondary structure distort as the tissue is loaded. These structural changes are followed as perturbations to tissue spectra. We load tissue in a custom-made dynamic mechanical tester that fits on the stage of a Raman microprobe and can accept hydrated tissue specimens. As the specimen is loaded in tension and/or compression, the shifts in mineral P-O₄ v₁ and relative band heights in the Amide III band envelope are followed with the microprobe. Average load is measured using a load cell while the tissue is loaded under displacement control. Changes occur in both the mineral and matrix components of bone as a response to elastic deformation. We propose that the mineral apatitic crystal lattice is deformed by movement of calcium and other ions. The matrix is proposed to respond by deformation of the collagen backbone. Raman microspectroscopy shows that bone mineral is not a passive contributor to tissue strength. The mineral active response to loading may function as a local energy storage and dissipation mechanism, thus helping to protect tissue from catastrophic damage.

Raman spectral imaging provides the means to study spatially localized response to controlled external perturbations to tissue specimens with light microscopy resolution. We discuss use of heparin-acrylic microbeads soaked with the protein fibroblast growth factor-2 (FGF2). Microbeads containing FGF2 are placed in murine calvarial tissue and stimulate abnormally rapid mineralization. The tissue response simulated the effects of craniosynostosis, a birth defect occurring in 1 in 2400 live births. We describe Raman imaging measurements of the spatial distribution of apatitic mineral and matrix (predominantly type I collagen) from normal murine calvarial tissue and murine calvarial tissue modeling craniosynostosis. We also discuss spectroscopic evaluation of the state of the mineral induced by the FGF2 beads.

We propose a waveguide based surface plasmon resonance optical biosensor for portable applications. Numerical results show that direct integration of a wire-grid polarizer is not only difficult, but also has poor polarization filtering performance. Our initial efforts focus on a waveguide based structure with prism coupling and the use of baculovirus as model pathogen.

Accurate real-time methods for the detection of pathogenic microorganisms in the agri-food industry would represent an improvement over standard methods of analysis. We are currently developing a non-contact, scanning optical system for the detection of bacteria on meat surfaces based on fluorescence lifetime and intensity measurements. The system detects autofluorescent light emitted by the naturally occurring fluorophores in bacteria. Potential expected advantages of this system include accurate and efficient 2D real-time mapping of bacterial contamination of surfaces, and elimination of sample-to-sample cross-contamination. Furthermore, as the technique only requires minimal sample preparation and handling, the chemical properties of the specimen are preserved. This article presents the preliminary results obtained from a time-resolved fluorescence imaging system for the characterization of a non-pathogenic gram-negative bacteria, Pseudomonas fluorescens. Additionally we present a particular application of the system of interest to the agri-food industry, demonstrating its potential as a real-time macroscopic imaging system for mapping bacterial contamination on meat surfaces. Initial results indicate that the combination of fluorescence lifetime and intensity measurements provides a means for characterizing biological media and for detecting microorganisms on surfaces.

The odorant-binding proteins (OBPs) are abundant low-molecular weight soluble proteins, which are secreted by the olfactory epithelium in the nasal mucus of vertebrates. These protein reversibly bind odorants with dissociation constants in the micromolar range. For this reason, they are good candidates as biological elements in the development of biosensors. Vertebrate OBPs belong to the lipocalin superfamily. Even if the members of this superfamily display low sequence similarity, all of them show a conserved folding pattern, that is an 8-stranded β-barrel flanked by an α-helix at the C-terminal end of the protein chain. The β-barrel defines a central apolar cavity, called calyx, whose role is to bind and transport hydrophobic odorant molecules. The detection of hazard exposure is becoming a priority in the third millennium, and OBPs are good candidates for detecting traces of explosive molecules in different environments such as luggage's storage rooms and public places. In this context, the measurement of refractive index of odor-binding protein in absence and in presence of odorant molecules have been performed in order to assess its usefulness as a probe for detection of hazardous agents. The work is instrumental to explore the possibility to realize a biosensor where the concentration of searched for substances is analyzed as a variation of the protein refractive index by means of suitable optoelectronic devices.

A sensitive biosensor (cytosensor) has been developed based on color changes in the toxin-sensitive colored living cells of fish. These chromatophores are highly sensitive to the presence of many known and unknown toxins produced by microbial pathogens and undergo visible color changes in a dose-dependent manner. The chromatophores are immobilized and maintained in a viable state while potential pathogens multiply and fish cell-microbe interactions are monitored. Low power LED lighting is used to illuminate the chromatophores which are magnified using standard optical lenses and imaged onto a CCD array. Reaction to toxins is detected by observing changes is the total area of color in the cells. These fish chromatophores are quite sensitive to cholera toxin, Staphococcus alpha toxin, and Bordatella pertussis toxin. Numerous other toxic chemical and biological agents besides bacterial toxins also cause readily detectable color effects in chromatophores. The ability of the chromatophore cell-based biosensor to distinguish between different bacterial pathogens was examined. Toxin producing strains of Salmonella enteritis, Vibrio parahaemolyticus, and Bacillus cereus induced movement of pigmented organelles in the chromatophore cells and this movement was measured by changes in the optical density over time. Each bacterial pathogen elicited this measurable response in a distinctive and signature fashion. These results suggest a chromatophore cell-based biosensor assay may be applicable for the detection and identification of virulence activities associated with certain air-, food-, and water-borne bacterial pathogens.

We describe the development of a novel generic approach to fluorescence sensing based on metal-enhanced fluorescence (MEF). This work follows our initial reports of radiative decay engineering (RDE), where we experimentally demonstrated dramatic signal enhancements of fluorophores positioned close to surface-bound silver nanostructures. The attractive changes in spectral properties of fluorophores includes increased rates of excitation, increased quantum yields, decreased fluorescence lifetimes with an increased photostability, and drastically increased rates of multi-photon excitation. In this report we present a new class of fluorescent biomarkers which are strongly enhanced by metallic particles. This has afforded the development of a novel generic approach for ultra-sensitive fluorescence assay technology. The assay platform utilizes metal particles deposited on glass/quartz surfaces, covered with sub-nanometer layers of a fluorescent biomarker. As such the fluorescence signal of the composite is strongly enhanced. This readily allows easy, quantitative and inexpensive fluorescence detection of minimal traces of specific antigens. We also explore different sensing geometries, such as using evanescent wave excitation.

We described a new approach to measuring DNA hybridization using surface plasmon-coupled emission (SPCE). This phenomenon occurs for fluorophores within few hundreds of nanometers of a thin metal film on a glass substrate, in our case a 50 nm thick silver film. Excited fluorophores coupled with the surface plasmons in the metal resulting in directional emission through the glass substrate. We studied the emission of Cy3-labeled DNA oligomers bound to complementary unlabeled biotinylated-oligomers, which were bound to the metal surface via a streptavidin-BSA monolayer. Hybridization resulted in directional emission of Cy3-DNA into the prism. Additionally, the use of SPCE resulted in suppression of interfering emission from non-complementary Cy5-DNA oligomers due to weaker coupling of the more distant fluorophores with the surface plasmons. A large fraction of the total potential emission can couple to the surface plasmon resulting in improved sensitivity. We expect SPCE to have numerous applications to nucleic acid analyses.

The fluorescence spectra from Φ6 and Φ12 cystoviruses and their pseudomonad host were investigated. The predominant fluorophore in both the Φ6 and Φ12 viruses and pseudomonas syringae is tryptophan. The emission maxima of the virus emission was found to be at 330 nm, compared to an emission maxima from the bacteria, which typically varies between 337 and 349 nm, depending on growth conditions. This difference Stokes shifts between the viruses and syringae hosts are most likely due to the virus proteins being in a much less polar environment than the bacteria proteins. This difference in Stokes shift was used to monitor the infection process.

Tiopronin-monolayer protected silver nanoparticles (average diameter = 5 nm) were partially replaced by thiolate boronic acid via ligand exchange. The uncapped boronic acids could be coupled with both a polysaccharide (Dextran) and a monoaccharide (glucose). Boronic acids capped on the particle were coupled selectively only by Dextran. With the decrease of charge on particle surface at pH = 5, boronic acids capped on the particle could be coupled with glucose. The particles were observed to aggregate along the Dextran chain, resulting in an absorbance decrease of the transverse plasmon at 397 nm and simultaneous rising of longitudinal plasmon at 640 nm. The aggregation increased with the concentration of Dextran. Luminescence intensity of phenyl boronic acid was enhanced by an upward deviation from a line with the concentration of Dextran in solution but the decay time was decreased, principally by the aggregation of the metallic cores.

Surface-enhanced Raman scattering (SERS) spectra of chemical agent simulants such as dimethyl methylphonate (DMMP), pinacolyl methylphosphonate (PMP), diethyl phosphoramidate (DEPA), and 2-chloroethyl ethylsulfide (CEES), and biological agent simulants such as bacillus globigii (BG), erwinia herbicola (EH), and bacillus thuringiensis (BT) were obtained from silver oxide film-deposited substrates. Thin AgO films ranging in thickness from 50 nm to 250 nm were produced by chemical bath deposition onto glass slides. Further Raman intensity enhancements were noticed in UV irradiated surfaces due to photo-induced Ag nanocluster formation, which may provide a possible route to producing highly useful plasmonic sensors for the detection of chemical and biological agents upon visible light illumination.

Alzheimer's disease is one of the most common forms of dementia, and causes steady memory loss and mental regression. It is also accompanied by severe atrophy of the brain. However, the pathological biomarkers of the disease can only be confirmed and examined upon the death of the patient. A commercial (Renishaw PLC, UK) Raman system with an 830 nm NIR diode laser was used to analyse brain samples, which were flash frozen at post-mortem. Ethical approval was sought for these samples. The Alzheimer's diseased samples contained a number of biomarkers, including neuritic plaques and tangles. The Raman spectra were examined by order to differentiate between normal and Alzheimer's diseased brain tissues. Preliminary results indicate that Alzheimer's diseased tissues can be differentiated from control tissues using Raman spectroscopy. The Raman spectra differ in terms of peak intensity, and the presence of a stronger amide I band in the 1667 cm^-1 region which occurs more prominently in the Alzheimer's diseased tissue. These preliminary results indicate that the beta-amyloid protein originating from neuritic plaques can be identified with Raman spectroscopy.

The ability of infrared (IR) spectroscopy to distinguish and map cancerous and non-cancerous tissue has opened the question of the origin of spectral differences between normal and cancerous cells. In this contribution, we report IR spectral maps of individual dried cancer cells, some of them in the process of cell division (mitosis), IR spectra of cells suspended in growth medium, and preliminary results of a statistical analysis of thousands of individual dried cancer cells.

In our former studies a diagnostic approach for the detection of transmissible spongiform encephalopaties (TSE) based on FT-IR spectroscopy in combination with artificial neural networks was described, based on a controlled animal study with terminally ill Syrian hamsters and control animals. As a consequence of the bovine spongiform encephalopathy (BSE) crisis in Europe, the development of a disgnostic ante mortem test for cattle has become a matter of great scientific importance and public interest. Since 1986 more than 180,000 clinical cases of BSE have been observed in the UK alone. Most of these cases were confirmed by post mortem examination of brain tissue. However, BSE-related risk assessment and risk-management would greatly benefit from ante mortem testing on living animals. For example, a serum-based test could allow for screening of the cattle population, thus, even a BSE eradication program would be conceivable. Here we report on a novel method for ante mortem BSE testing, which combines infrared spectroscopy of serum samples with multivariate pattern recognition analysis. A classification algorithm was trained using infrared spectra of sera from more than 800 animals from a field study (including BSE positive, healthy controls and animals suffering from viral or bacterial infections). In two validation studies sensitivities of 85% and 87% and specificities of 84% and 91% were achieved, respectively. The combination of classification algorithms increased sensitivity and specificity to 96% and 92%, respectively.