Candida albicans is an opportunistic pathogen, generally though to be of endogenous origin, with however reported outbreaks. Epidemilogy of C. albicans has been studied so far by genotypic methods mainly, including the classical RAPD analysis. Albeit powerful, genotypic techniques are expensive, time consuming and complex to implement. FTIR spectroscopy is simple, rapid, inexpensive and an increasingly used technique for the identification of microorganisms. As a phenotypic method, it provides rapid whole cells 'fingerprinting' using few consumables and can detect very subtle differences between strains of the same species. In this study, C. albicans strains isolated from 50 patients from six hospital units were collected and studied by FTIR spectroscopy and RAPD-PCR. Discrimination of strains was computed using classification algorithms on selected features of the spectral data. Results from 10 patients, for whom iterative sampling was possible, are presented and discussed. Emphasis was laid on the reproducibility of dat for strain-level identification. FTIR analysis shows that (a) the C. albicans spectra were different from one patient to another, (b) seven patients exhibit each a homogeneous group while three patients display each two groups of strains. RAPD-PCR and FTIR analyses correlate quite well showing that FTIR spectroscopy could be a potential epidemiological tool in the control of nosocomial fungal infections.

Transmissible spongiform encephalopathies (TSE), such as BSE in cattle, scrapie in sheep and goats, and Creutzfeldt-Jakob disease in man are a group of fatal infectious diseases of the central nervous system that are far from being fully understood. Presuming the pathological changes to originate from small disease-specific compositional and structural modifications at the molecular level, Fourier-transform infrared (FTIR) spectroscopy can be used to achieve insight into biochemical parameters underlying pathogenesis. We have developed an FTIR microspectroscopy-based strategy which, as a combination of image reconstruction and multivariate pattern recognition methods, permitted the comparison of identical substructures in the cerebellum of healthy and TSE-infected Syrian hamsters in the terminal stage of the disease. Here we present FTIR data about the pathological changes of scrapie-infected and normal tissue of the gray matter structures stratum granulosum and stratum moleculare. IR spectroscopy was also applied to tissue pieces of the medulla oblongata of infected and control Syrian hamsters. Mapping data were analyzed with cluster analysis and imaging methods. We found variations in the spectra of the infected tissue, which are due to changes in carbohydrates, nucleic acids, phospholipids, and proteins.

Bone tissue consists of a carbonated apatite-like mineral supported on a hydrated, collagen-rich protein matrix. Despite extensive studies into the macroscopic characteristics of bone, much about the early stages of bone formation remains unknown. Raman microspectroscopy and imaging are increasingly important tools for the study of mineralized tissue, due to advancements in both spectral acquisition and analysis protocols. With this technique, mapping of both organic and inorganic components of bone, in addition to determining their distributions with high spatial resolution across a specimen, can be realized. We have employed Raman microscopy to investigate the early stages of mineralization in four different mouse calvarial systems: typical and atypical osteoblastic (bone forming) cell cultures and healthy and diseased bone tissue. These systems are commonly utilized as models for mineralization. The mineral deposited by osteoblast cultures grown atypically gives a Raman signal completely different to that observed in osteoblast cultures grown in the conventional manner. Similarly, Raman images of healthy and diseased bone tissue show differences in the relationship of the mineral and matrix environments. In this report, we compare the several differences between these four mineral environments, and discuss the chemistry of mineral maturation observed.

Healing is important for the success of the insertion of implants and for treating traumatic or pathologic injuries of the bone. Lasertherapy has been suggested as a mean of improving bone healing. Raman spectroscopy was used to assess the amount of both inorganic and organic components of irradiated and non-irradiated bone around dental implants inserted in to the tibia. Fourteen rabbits received a titanium implant on the tibia, eight of them were irradiated with 830nm laser and six acted as controls. The animals were sacrificed 15, 30 and 45 days after the surgery, and specimens were prepared for Raman spectroscopy, which was collected at every four points from each three thirds of the bone around the implants. The results showed significant differences in the concentration of inorganic components in irradiated specimens between 15 and 30days, 15 and 45; between irradiated and controls 30 and 45 days after surgery. Concentration of organic components was also significantly different between irradiated and controls in periods of 30 to 45 days after surgery. It is concluded that LLLT does improve bone healing and this can be safely assessed by Raman Spectroscopy.

The skeleton is certainly essential to our physiology. Yet, surprisingly little is understood about how bone responds when a load is applied. This is particularly true at the ultrastructural level, where neither the behavior under stress of the collagenous matrix nor that of the calcium phosphate mineral has been explored. Most standard techniques used in the investigation of the biomechanical properties of bone tissue are unable to obtain information at this level. Raman spectroscopy, however, is capable of probing biological specimens at the molecular level, without interference from water or common histological stains. Using this technique, we are able to observe changes in both the mineral and the matrix of bone during and after the application of load. These changes manifest themselves as band shifts in the bone Raman spectrum. We utilize this ability in this study. Two-dimensional Raman imaging is used to investigate the effect of macroscopic fracture on the mineral in sections of femora from 4-month-old and 18-month-old mice. Changes are seen in the mineral around fractured areas that indicate the fracture process may be causing a phase transformation in the bone mineral, similar to the 'transformation toughening' process that is observed in many ceramics. We discuss the physiological implications of these results.

The vicious circle of insulin resistance and hyperinsulinemia is considered to precede the manifestation of diabetes type-2 by decades and the corresponding cluster of risk factors is described as the 'insulin resistance syndrome' or 'metabolic syndrome'. Since the present diagnosis of insulin resistance is expensive, time consuming and cumbersome, there is a need for diagnostic alternatives. We conducted a clinical study on 129 healthy volunteers and 99 patients suffering from the metabolic syndrome. We applied mid-infrared spectroscopy to dried serum samples from these donors and evaluated the spectra by means of disease pattern recognition (DPR). Substantial differences were found between the spectra originating from healthy volunteers and those spectra originating from patients with the metabolic syndrome. A linear discriminant analysis was performed using approximately one half of the sample set for teaching the classification algorithm. Within this teaching set, a classification sensitivity and specificity of 84 percent and 81 percent respectively can be derived. Furthermore, the resulting discriminant function was applied to an independent validation of the remaining half of the samples. For the discrimination between 'healthy' and 'metabolic syndrome' a sensitivity and a specificity of 80 percent and 82 percent respectively is obtained upon validating the algorithm with the independent validation set.

The objective of this work was to use infrared (IR) fiberoptic spectroscopy for the analysis of urinary salts. Urine samples were obtained (with no sample preparation) from two groups of patients: 24 stone forming patients, after shock wave lithotripsy, and 24 normal subjects of similar ages. IR absorption measurements were performed in real time, using Fiberoptic Evanescent Wave Spectroscopy system, based on IR transmitting silver halide fibers. The absorption data were compared with the IR spectra of aqueous solutions with known concentrations of known urinary salts. The results were then used for the study of the chemical composition of salts in urine samples and for a quantitative analysis of the concentration of these salts. We established the composition of the stones in 20 of the 24 stone forming patients, based on the characteristic absorption peaks for oxalates, carbonates, urates and phosphates observed in their urinary samples. We also determined the concentrations of these salts in the urine samples with average error of 20 percent.

We devoted efforts to develop an analytical method of plasma able to provide both a high sensitivity and a global overview of its biomolecular contents along with the variations of these ones. Among candidates, transmittance FT-IR spectrometry has proved to be highly efficient. It has been used to analyze plasma micro samples using an iterative process. Results in accordance with clinical data were obtained from a single FT-IR spectrum for the following biomolecules: amino-acids, fatty acids, albumin, glucose, fibrinogen, lactate, triglycerides, glycerol, urea, (alpha) ₁-antitrypsin, alpha₂-macroglobulin, transferin, Apo-A₁, Apo-B, Apo-C₃, IgA, IgD, IgG₁, IgG₂, IgG₃, IgG₄, IgM, haptoglobin, and (alpha) ₁-acid glycoprotein. Moreover, cholesterol contribution may be determined on the same IR spectrum. Therefore, as only micro samples are necessary, high frequency blood analysis become available. This method was used to monitor inflammatory processes related to given metabolic stresses. Moreover, FT-IR spectrum constitutes a 'metabolic photography' of the subject, allowing classification between metabolic groups (pathologic or others). It was used on difference spectra in order to raise 'signal to noise' ratio by elimination of the unvarying spectral contribution. Among others, it allowed to uncover overtraining in high-level sportsmen several weeks before any physiologic or clinical symptom occurred.

Near-infrared spectroscopic methods have been developed to determine the degree of hydration of human skin in vivo. Reflectance spectroscopic imaging was used to investigate the distribution of skin moisture as a function of location. A human study in a clinical setting has generated quantitative data showing the effects of a drying agent and a moisturizer on delineated regions of the forearms of eight volunteers. Two digital imaging systems equipped with liquid-crystal tunable filters were used to collect stacks of monochromatic images at 10-nm intervals over the wavelength bands 650-1050 nm and 960-1700 nm. Images generated from measurements of water absorption-band areas at three different near-IR wavelengths (970, 1200, and 1450 nm) showed obvious differences in the apparent distribution of water in skin. Changes resulting from the skin treatments were much more evident in the 1200-nm and 1450-nm images than in the 970-nm ones. The variable sensitivity of the method at different wavelengths has been interpreted as being the result of different penetration depths of the infrared light used in the reflectance studies. Ex-vivo experiments with pigskin have provided evidence supporting the relationship between wavelength and penetration depth. Combining the hydration results from several near-IR water bands allows additional information on hydration depth to be obtained.

Fourier Transform Infrared (FTIR) spectroscopic systems make use of Attenuated Total Reflection (ATR) elements for the study of skin in dermatology. FTIR - ATR allows real time and reagent-less analysis of several components, simultaneously. The potential for skin studies is increased by the development of the flexible fiber optic sensor made from infrared transparent polycrystalline silver halide. Segments of fibers can replace the ATR sensing elements inside an FTIR system. Moreover a Fiberoptic Evanescent Wave Spectroscopy (FEWS) can also be used for real time in vivo measurement on skin, in situ. We used FEWS to study the diffusion of UV sunscreen lotions from the outer skin layer into the dermis and epidermis, and used the various absorption bands to differentiate between the behavior of the organic and the water molecules in the lotion. FEWS can be a powerful tool for studying the transport of drugs and cosmetic creams through the skin from the stratum corneum to the dermis and epidermis and for studying the lateral diffusion of various molecules into the skin, in vivo and in real time.

Chemical Factor Analysis (CFA) algorithms were applied to transform complex Fourier transform infrared fiberoptical evanescent wave (FTIR-FEW) normal and malignant skin tissue spectra into factor spaces for analysis and classification. The factor space approach classified melanoma beyond prior pathological classifications related to specific biochemical alterations to health states in cluster diagrams allowing diagnosis with more biochemical specificity, resolving biochemical component spectra and employing health state eigenvector angular configurations as disease state sensors. This study demonstrated a wealth of new information from in vivo FTIR-FEW spectral tissue data, without extensive a priori information or clinically invasive procedures. In particular, we employed a variety of methods used in CFA to select the rank of spectroscopic data sets of normal benign and cancerous skin tissue. We used the Malinowski indicator function (IND), significance level and F-Tests to rank our data matrices. Normal skin tissue, melanoma and benign tumors were modeled by four, two and seven principal abstract factors, respectively. We also showed that the spectrum of the first eigenvalue was equivalent to the mean spectrum. The graphical depiction of angular disparities between the first abstract factors can be adopted as a new way to characterize and diagnose melanoma cancer.

Infrared absorption of two pairs of non-tumorigenic and tumorigenic cells suspended in phosphate buffered saline have been measured. Suspensions of cells with several different growth cycle distributions were measured. The effect of both growth cycle and tumorigenity on the infrared absorption spectrum will be presented. For example, changes in absorption in the phosphate absorption region were observed for suspensions with different cell cycle distributions. We will discuss the biochemistry which may cause these changes. We have found that spectra of isolated nuclei allow the DNA spectra to be studied, without the confounding influence of RNA. Therefore, the measurement of isolated nuclei may represent a method of detecting changes in DNA architecture with cell cycle. As part of this exploratory study we have also examined the variation in spectra with cell type and compared epithelial cells with fibroblast cells. Very little change was observed. Similarly we saw very little change in the spectra of tumorigenic and non-tumorigenic cells harvested with similar cell cycle distributions. Changes in the spectra were observed when rapidly growing tumorigenic cells were compared to slowly replicating nontumorigenic cells.

The prospect of utilising NIR-Raman spectroscopy for analysis of gastro-intestinal (GI) tissue has been explored both with snap-frozen and formalin fixed samples. In the oesophagus large sample numbers have been employed and the full spectrum of pathology has been studied. Multivariate analysis techniques have been employed to optimally separate the groups and spectral diagnostic models have been constructed and evaluated by employing cross-validation testing. Sensitivities have been shown to vary between 73 and 100 percent and specificities between 91 and 100 percent, depending on pathology group and tissue type.

A new approach is presented to distinguish cancerous from normal brain tissue via linear discriminant analysis of Fourier transform infrared (FTIR) spectra. FTIR microspectroscopy was used to map various thin-section tumor samples with different malignancy grades (grades II-VI) and non-tumor samples obtained from various patients by surgical removal. Spectral analysis revealed features characteristic of tumors with increasing malignancy. A genetic region selection algorithm combined with linear discriminant analysis was used to derive classifiers distinguishing among spectra of control tissue, astrocytoma grade II, astrocytoma grade III and glioblastoma grade IV. Employing the World Health Organization histopathological diagnostic scheme as the gold standard, the spectra were classified with a success rate of approximately 85 percent. These results demonstrate the potential of the combination of FTIR spectroscopy and pattern recognition routines in providing a more objective method for brain tumour grading and diagnosis.

Immunohistochemistry contributes to determine the site of origin of the tumor, helps setting the treatment and the prognosis. However, these assays are both time consuming when treatment may be an emergency, and expensive. Instead of the usual biochemistry we are using for the first time for medical issues Fourier-transform IR ellipsometry, a non- invasive optical characterization technique used in the semiconductor field to characterize bare substrates and thin films. Working in the mid-IR makes this technique sensitive to the molecules present inside the cell. Since the ellipsometric analysis can be performed on tissue slides extended on a specular surface, they may provide the physician with a chemical map of the sample. Here we show that ellipsometry can detect and measure physiological concentration of glycogen diluted in water and retrieve the polysaccharide band in a tissue slide. Our objective is to show that ellipsometry can give a quick and comprehensive analysis of the diverse biological markers forming the living matter.

Optical spectroscopic detection of early malignancy is becoming more widely accepted in academic circles, however much work remains to be done before full recognition by the medical community is achieved. The majority of published studies to date have demonstrated the potential of optical diagnosis techniques using small sample numbers in a selected patient population. Many are completed without a solid understanding of the shortcomings of histopathology, the 'gold standard' for cancer detection. For the development of a new technique to improve diagnosis it is vital that more rigorous protocols are employed in large-scale clinical trials. The prospect of utilizing NIR-Raman spectroscopy for the analysis of neoplastic gastrointestinal tissue has been thoroughly explored by a multi-disciplinary team including surgeons, pathologists, and spectroscopists. This study demonstrates the need for rigorous experimental protocols and histopathological analysis by a panel of expert pathologists. Measurements of tissue specimens from nine different pathological groups describing the full spectrum of disease in the oesophagus have been made. Only homogeneous samples with consensus pathology opinion were used to construct a training data set of Raman spectra. Models were constructed using multivariate analysis techniques and tested using cross-validation.

Hyperspectral (HS) imaging has been useful in the monitoring of several medical conditions, which to date have generally involved changes in skin oxygenation in isolated regions of interest such as skin flaps or small burns. Here, by contrast, we present a study in which HSI was used to assess the cutaneous manifestations of significant systemic events. HS imaging of the ventral surface of the lower jaw was used to monitor changes in skin oxygenation during hypovolemic shock induced by pulmonary contusion and hemorrhage in a porcine model, and to monitor the subsequent recovery of oxygenation following resuscitation. Changes are seen both quantitatively, in the level of skin oxygenation as determined by the fitting of reference hemoglobin and deoxyhemoglobin spectra to sample spectra, and qualitatively, in the observed spatial distribution or pattern of oxygenation-related changes in the skin. Linear regression was used to correlate these changes with invasively obtained parameters to include mixed venous oxygen saturation and systemic arterial blood pressure. Historically, the assessment of skin color and mottling has been an important, albeit inexact, component of resuscitation algorithms. Now, it is possible to analyze these variables during shock and resuscitation in an objective manner. The clinical utility of these advances needs to be determined.

Raman spectroscopy has been shown to have the potential for providing differential diagnosis in the cervix with high sensitivity and specificity in previous in vitro and in vivo studies. A clinical study was designed at Vanderbilt University Medical Center to further evaluate the potential of near IR Raman spectroscopy for in vivo detection of squamous intra-epithelial neoplasia, a pre-cursor to cervical cancer, in a clinical setting. In this pilot in vivo clinical study, using a portable system, Raman spectra are collected using clinically feasible integration times during colposcopic evaluation. Multiple Raman spectra were acquired form colposcopically normal and abnormal sites prior to excision of tissue from patients with known abnormalities of the cervix. Measured Raman spectra were processed for nosie and background fluorescence using novel signal processing techniques. The resulting spectra were correlated with the corresponding histological diagnosis to determine empirical differences in spectra between different diagnostic categories. Using histology as the gold standard, multivariate statistical techniques were also used to develop discrimination algorithms with the hopes of developing this technique into a real time, non-invasive diagnostic tool.

Hemoglobin (Hb) within single erythrocytes (red blood cells), adsorbed on poly-lysine coated glass surfaces, was studied using resonance Raman spectroscopy and global Raman imaging. The erythrocytes were found to be sensitive to both surface adsorption and to the laser light. Topological changes of the cell membrane were observed immediately after cell adsorption in Raman images. We observed a photo-induced increase of the fluorescence background occurring simultaneously with a decrease in the Hb Raman signal. Concurrent changes in Raman spectra revealed a conversion of oxy-Hb to the met-Hb state. However, at a low accumulated photon dose, the preparation method enabled the recording of Raman spectra during the oxygenation cycle of a single red blood cell in buffer, which shows that Hb was in an in-vivo environment. Thus, Raman spectroscopy of functional Hb in isolated red blood cells is feasible.