Infrared transmission/absorption measurements of cells and biofluids in water are restricted to very short optical pathlengths. When the amide I and amide II bands of protein constituents have to be analysed, path-lengths of less than 8 μm are necessary. Infrared spectra of cancer cells were collected from physiological buffer solutions utilizing custom-made mid-infrared compatible IR-cuvettes. The technology permitted to obtain cell-type specific spectral signatures and probe biochemical changes induced by varying temperatures or cell-drug interaction. Optical path-lengths of 8-30 μm were used on a set of microbial test strains to evaluate, whether the methodology can also be used to discriminate and identify micro-organisms. A semi-automatic methodology was developed for the analysis of liquid serum samples, which combines simple sample handling with high sample throughput and extreme measurement reproducibility. The applicability of this infrared technology to the analysis of liquid serum samples from cattle and human beings suffering from various acute viral or bacterial infections was explored testing the interrelationship between α-helical and β-sheet specific spectral signatures in the amide I band contour and total albumin and globulin content in serum. The technical details, advantages, and limitations of the new technology are described in the context of developing a routine, IR-based biodiagnostic technique for biofluids and biological cells.

The determination of protein structure and function plays an important role in biomedical and biochemical research. Common techniques that give full structural information do not permit in-vivo measurements. Infrared spectroscopy has a sufficient sensitivity to examine the structure of proteins solution under in-situ conditions and even on surfaces. However, measurements at few spots on the surface are not suitable to find out the blood compatibility of the protein layer, because the changes in conformation occur often in small domains. Here we report on the investigations of adsorbed fibrinogen with FTIR imaging. FTIR imaging permits an identification of coagulation spots in the micrometer range and the identification of coagulation spots on the implant material.

For clinical research, in-vivo blood glucose monitoring is an ongoing important topic to improve glycemic control in patients with non-adequate blood glucose regulation. Critically ill patients received much interest, since the intensive insulin therapy treatment, as established for diabetics, reduces mortality significantly. Despite the existence of commercially available, mainly amperometric biosensors, continued interest is in infrared spectroscopic techniques for reagent-free glucose monitoring. For stable long-term operation, avoiding also sensor recalibration, a bed-side device coupled to a micro-dialysis probe was developed for quasi-continuous glucose monitoring. Multivariate calibration is required for glucose concentration prediction due to the complex composition of dialysates from interstitial body fluid. Measurements were carried out with different test persons, each experiment lasting for more than 8 hours. Owing to low dialysis recovery rates, glucose concentrations in the dialysates were between 0.83 and 4.44 mM. Standard errors of prediction (SEP) obtained with Partial Least Squares (PLS) calibration and different cross-validation strategies were mainly between 0.13 and 0.18 mM based on either full interval data or specially selected spectral variables.

Mid-infrared and Raman spectroscopy together with multivariate data analysis offers the potential to be applied to clinical laboratory analysis due to their reagent-free nature, the speed of analysis and the possibility of obtaining a variety of information from a single measurement. In what we believe to be among the largest studies on mid-infrared and Raman spectroscopy for the analysis of multiple analytes in serum, samples from 247 donors have been analyzed with the emphasis on reproducibility. In an independent validation, root-mean-square errors of prediction (RMSEP) ranged from 328 mg/dL for the quantification of protein (mean concentration: 7008 mg/dL) using mid-infrared spectroscopy to 1.1 mg/dL for uric acid (mean concentration: 5.3 mg/dL) in the case of Raman spectroscopy. Both techniques deliver similar performances. We also performed first steps towards determining system precision and accuracy. In a fivefold measurement of 5 randomly chosen samples from this study, precision and accuracy range from 4% to 16% and from 4% to 29%, respectively. However, when considering the physiological and pathological range of concentrations of analytes, vibrational spectroscopy might open the path towards less expensive and more rapid multiparameter analysis of small sample volumes in those cases, in which moderate accuracy is permissible.

We have refined of our previously published tissue modulation technique for obtaining Raman spectra of blood in fingertip capillary beds. Results from the newest LighTouch device benefit from more consistent management of applied force and temperature and more consistent tissue placement. Comparing these more precisely obtained spectra with other spectra obtained from the same capillary beds using the natural heart driven pulse as modulation reveals essential aspects of microcirculation such as plasma skimming, the Faraeus effect and the Faraeus-Lindqvist effect. We discuss these results in the context of performing noninvasive quantitative analysis of blood and blood components in vivo. We show the first Raman spectra of human blood plasma noninvasive, in vivo.

Only few studies have attempted to characterize biological materials by THz spectroscopy. Most of these used either solid samples or biological tissues. In this work, we present results of THz spectroscopic characterization of dilute solutions of DNA samples. Water and heavy water (D₂O) have strong absorbance that overlap significantly with important absorption bands of biomolecules in conventional FTIR spectroscopy. Cumbersome spectral subtractions and highly concentrated samples are therefore required to partially overcome problems of water interference in FTIR spectra of biomolecules. Although liquid water absorbs and contributes to background in the THz spectral range of interest, the level of water absorption in the low THz range is at least 2.5 orders of magnitude less than in the far IR. Here, we demonstrate that reproducible spectra of dilute solutions of DNA in the frequency range 10-24 cm^-1 can be obtained. We show that dilute aqueous samples of DNA produce THz spectra with signals that do not overlap with those of water. This is a significant achievement towards the goal of developing THz resonance spectroscopy as a useful tool for the biological sciences because all biological functions of DNA and proteins take place in aqueous environments. A simple technique for sample preparation and characterization is described. Samples containing as little as 100 ng of DNA in 10 μl of water (0.01 mg/ml or 0.001%) have been prepared and measured. The signal/noise ratios of THz spectra of these samples are sufficient to detect reproducible resonances at several characteristic frequencies. The effect of orientation on different substrates and the mechanism of sensitivity enhancement in these samples is discussed. It should be possible to extend these methods to also study proteins in dilute solutions. Advantages of using dilute samples include small quantities of biological material required, the absence of interference from interactions between neighboring molecules, and the absence of problems with light-scattering that are often encountered with short wave-length optical techniques.

Far field radiation pattern under tight focusing condition is investigated in Coherent Anti-stokes Raman Scattering (CARS) microscopy both in the forward (F-CARS) and backward (E-CARS) directions. While we assume no refraction index mismatch between the sample and the environing medium, our rigorous numerical electromagnetic computation takes into account the exact polarizations of the excitation laser beams and of the induced nonlinear dipoles. F-CARS and E-CARS radiation patterns, as well as their divergence, are studied as a function of the size of the sample object and compared to the excitation beams.

Infrared breath analysis is used in diagnostics of respiratory diseases, pulmonary function testing, and for metabolic studies. With selective and highly sensitive instruments exhaled trace gas concentrations can be related to specific diseases. For many applications also a time resolution below 0.1s is needed. Frequently, performance is limited by the IR source. New developments offer solutions even for compact instruments. Different setups employing quantum cascade lasers (QCL), VCSELs, and a new optically pumped IR emitter are compared focusing on CO₂ measurements as an example.

A fast and unambiguous identification of microorganisms is necessary not only for medical purposes but also in technical processes such as the production of pharmaceuticals. Conventional microbiological identification methods are based on the morphology and the ability of microbes to grow under different conditions on various cultivation media depending on their biochemical properties. These methods require pure cultures which need cultivation of at least 6 h but normally much longer. Recently also additional methods to identify bacteria are established e.g. mass spectroscopy, polymerase chain reaction (PCR), flow cytometry or fluorescence spectroscopy. Alternative approaches for the identification of microorganisms are vibrational spectroscopic techniques. With Raman spectroscopy a spectroscopic fingerprint of the microorganisms can be achieved. Using UV-resonance Raman spectroscopy (UVRR) macromolecules like DNA/RNA and proteins are resonantly enhanced. With an excitation wavelength of e.g. 244 nm it is possible to determine the ratio of guanine/cytosine to all DNA bases which allows a genotypic identification of microorganisms. The application of UVRR requires a large amount of microorganisms (> 10⁶ cells) e.g. at least a micro colony. For the analysis of single cells micro-Raman spectroscopy with an excitation wavelength of 532 nm can be used. Here, the obtained information is from all type of molecules inside the cells which lead to a chemotaxonomic identification. In this contribution we show how wavelength dependent Raman spectroscopy yields significant molecular information applicable for the identification of microorganisms on a single cell level.

Micro-Raman spectroscopy covering a frequency range from 200 to 4000 cm^-1 was used to image human skin melanocytes and keratinocytes with a spatial resolution of 0.5 μm. The cells were either cultivated on glass microscope slides or were located within thin sections of skin biopsies mounted on low fluorescence BaF₂. A commercially available system was used to obtain the spectra utilizing a x100 long working distance objective with a numerical aperture of 0.8, and a cooled CCD. Both 633 and 515 nm excitations were tried, although the latter proved to be more effcient at producing Raman emission mostly due to the 1/λ⁴ dependence in light scattering. Fluorescence emission from the cells was surprisingly low. The excitation power at the sample was kept below about 2 mW to avoid damaging the cells; this was the limiting factor on how quickly a Raman image could be obtained. Despite this diffculty we were able to obtain Raman images with rich information about the spectroscopic and structural features within the cytoplasm and cell nuclei. Differences were observed between the Raman images of normal and malignant cells. Spectra from purified DNA, RNA, lipids, proteins and melanin were obtained and these spectra were compared with the skin cell spectra with the aim of understanding how they are distributed over a cell and how the distribution changes between different cells.

A new class of Surface-Enhanced Raman Scattering (SERS) substrates have been engineered by exploiting both Photonic Crystal (PC) and semiconductor technologies. Gold coated inverted pyramids nanotextured substrates allow reproducibility <10% and enhancement factors > 10⁶ over large areas. Modelling and optical characterization of the engineered structures is demonstrated. Examples of applications to amino acids and illicit drug detection are given. Concentrations as low as ppm-ppb (mg/mL to ng/mL) have been measured depending on the adsorbed analytes. Information on structure and conformation of the molecule is inferred due to the richer nature of SERS spectra.

We report on the feasibility of surface enhanced Raman scattering (SERS) as a highly sensitive technique for detecting peptide phosphorylation. Compared with existing techniques for quantifying peptide phosphorylation, such as high-performance liquid chromatography (HPLC), the short scanning and processing time associated with SERS makes it an attractive alternative for measurement on a near-real-time basis at sub micro-molar concentrations. Using the recently reported drop-coating deposition Raman method we compare our SERS spectra to normal Raman spectra that would otherwise be unobtainable at such low concentration.

A novel application of surface-enhanced Raman spectroscopy (SERS) for in-vitro osteoarthritis (OA) biomarker detection is described. Hyaluronic acid (HA) is a potential OA biomarker and synovial fluid levels of HA have been correlated with progression of joint space narrowing. However, current immunoassay and chromatographic methods that identify HA in synovial fluid are cumbersome and often require sophisticated instrumentation. Raman spectroscopy may be an alternative to these analytical methods, providing rapid identification of HA using characteristic Raman bands. Yet, previous reports of un-enhanced Raman spectroscopy for hyaluronic acid are in aqueous solutions exceeding 1000X in-vivo concentrations because HA is a weakly scattering polysaccharide. Surface-enhanced Raman spectroscopy can improve detection limits by 100-1000 times and we present, to our best knowledge, the first surface-enhanced Raman spectra of hyaluronic acid. Moreover, the recent commercial availability of stable SERS gold substrates has enabled rapid SERS detection of this biomarker at concentrations diluted by more than an order of magnitude, compared to previous literature reports. Preliminary results of easily and rapidly observing hyaluronic acid at low concentrations in aqueous solutions supported further studies in synthetic models of biofluids, such as artificial synovial fluid, that contain HA at low concentrations. These complex fluids contain proteins that compete for the SERS-active sites on the substrate, and the resulting spectra are dominated by protein Raman bands. We apply a simple and validated protein precipitation protocol to artificial synovial fluid prior to deposition onto the SERS substrate. We find that HA is easily detected in these fluids after protein removal treatment.

The phagocyte NADPH oxidase is a crucial enzyme in the innate immune response of leukocytes against invading microorganisms. The superoxide (O₂^-) that is generated by this enzyme upon infection is directly and indirectly used in bacterial killing. The catalytic subunit of NADPH oxidase, the membrane-bound protein heterodimer flavocytochrome b₅₅₈, contains two heme moieties. Here, we first briefly discuss our recent confocal resonant Raman (RR) spectroscopy and microscopy experiments on flavocytochrome b₅₅₈ in both resting and phagocytosing neutrophilic granulocytes. Such experiments allow the determination of the redox state of flavocytochrome b₅₅₈ inside the cell, which directly reflects the electron transporting activity of NADPH oxidase. Subsequently, we report that incubation of murine RAW 264.7 macrophages with PolyActive microspheres for 1 week in culture medium leads to morphological and biochemical changes in the macrophages that are characteristic for the generation of macrophage-derived foam cells. Lipid-laden foam cells are the hallmark of early atherosclerotic lesions. Using nonresonant Raman spectroscopy and microscopy, we demonstrate that the numerous intracellular droplets in macrophages exposed to microspheres are rich in cholesteryl esters. The finding that phagocytic processes may trigger foam cell formation reinforces the current belief that (chronic) infection and inflammation are linked to the initiation and progression of atherosclerotic lesions. The study of such a connection may reveal new therapeutic targets for atherosclerosis treatment or prevention.

There is no universally accepted grading system for the classification of Ductal Carcinoma in Situ (DCIS) although the diagnosis of DCIS has increased (2-20%) with screening mammography. (1) At present there are more than six different classifications and grading systems. Infrared spectroscopy is a non-invasive, rapid and specific technique used to analyse biological tissue. Spectral analysis of the chemical fingerprint within the duct would reveal spectral differences according to absorption and transmission characteristics of different grades of DCIS. An existing model of histopathological classification which is locally accepted has been tested and evaluated in this study. 19 ducts from different biopsy specimens were marked on H&E stained sections by two breast pathologists, according to the locally accepted classification. A consecutive unstained 20μm section was subjected to infrared analysis (Perkin-Elmer). Principal component analysis was undertaken using Matlab. Pseudocolor maps of the principal component scores delineated morphological features of the ducts. Peaks in the corresponding principal component loads were identified to enable understanding of the biochemical changes associated with different grades of DCIS. A 4-group cross-validated classification model was developed using multivariate statistical analysis with selected spectra from different grades of DCIS. The classification model demonstrated good separation of the different grades of the DCIS with a sensitivity of 80-99% and specificity of 92-98%. Infrared spectroscopy is a highly sensitive and specific technique for the demonstration of biochemical changes within the proliferative duct. It could aid in reclassifying the grades of DCIS in accordance with the biochemical and morphological changes that occur with proliferation. Infrared spectroscopy has potential as an added tool for the pathologist to diagnose in vitro.

Within the next 50 years Alzheimer's disease is expected to affect 100 million people worldwide. The progressive decline in the mental health of the patient is caused by severe brain atrophy generated by the breakdown and aggregation of proteins, resulting in β-amyloid plaques and neurofibrillary tangles. The greatest challenge to Alzheimer's disease lies in the pursuit of an early and definitive diagnosis, in order that suitable treatment can be administered. At the present time, definitive diagnosis is restricted to post-mortem examination. Alzheimer's disease also remains without a long-term cure. This research demonstrates the potential role of Raman spectroscopy, combined with principle components analysis (PCA), as a diagnostic method. Analyses of ethically approved ex vivo post-mortem brain tissues (originating from frontal and occipital lobes) from control (3 normal elderly subjects and 3 Huntingdon's disease subjects) and Alzheimer's disease (12 subjects) brain sections, and a further set of 12 blinded samples are presented. Spectra originating from these tissues are highly reproducible, and initial results indicate a vital difference in protein content and conformation, relating to the abnormally high levels of aggregated proteins in the diseased tissues. Further examination of these spectra using PCA allows for the separation of control from diseased tissues. The validation of the PCA models using blinded samples also displays promise for the identification of Alzheimer's disease, in conjunction with secondary information regarding other brain diseases and dementias. These results provide a route for Raman spectroscopy as a possible non-invasive, non-destructive tool for the early diagnosis of Alzheimer's disease.

Early dental caries detection facilitates implementation of non-surgical methods for arresting caries progression and promoting tooth remineralization. We present a method based on Raman spectroscopy with near-IR laser excitation to provide biochemical contrast for detecting and characterizing incipient carious lesions found in extracted human teeth. Changes in Raman spectra are observed in PO₄^3- vibrations arising from hydroxyapatite of mineralized tooth tissue. Examination of various intensities of the PO₄^3- ν2, ν3, ν4 vibrations showed consistent increased intensities in spectra of carious lesions compared to sound enamel. The spectral changes are attributed to demineralization-induced alterations of enamel crystallite morphology and/or orientation. This hypothesis is supported by reduced Raman polarization anisotropy derived from polarized Raman spectra of carious lesions. Polarized Raman spectral imaging of carious lesions found on whole (i.e. un-sectioned) tooth samples will also be presented.

The stratum corneum (SC) water concentration gradient is fundamental to skin's role as a barrier, regulating its physical and biochemical properties. Standard instruments utilizing changes in SC electrical properties to estimate SC water concentration provide simple, rapid measurements but cannot provide true interval data as a function of depth. Confocal Raman spectroscopy (CRS) of human subjects provides non-invasive, real-time, in vivo measures of molecular concentration profiles. A state-of-the-art confocal Raman microspectrometer equipped with a fiber-coupled laser source operating at a wavelength of 671 nm was used to obtain measurements in the high wavenumber region (~2400-4000 cm^-1). An aircooled, high-sensitivity back-illuminated, deep-depletion CCD camera captured radiation scattered inelastically from focal planes within the skin in vivo (a high-precision, computer-controlled piezo-electric stage and objective allowing depth resolutions of <5 μm, with over-sampling). High-wavenumber data were analyzed to provide semi-quantitative measures of water concentration ([water] / [protein + water]) across the SC. This new technique was used to study changes in SC water concentration gradients in human skin in vivo, in response to treatment with topical moisturizing products. The results of a blinded, randomized 3-week study in human volunteers will be presented, in particular, the significant, unique effects of a topical moisturizer containing niacinamide on SC water concentration gradient, as measured by CRS, in vivo. The approach to compare SC water gradient effects will be discussed and the utility of this exciting new method will be compared and contrasted to existing methodology.

We demonstrate the first transcutaneous Raman spectroscopic measurements of bone tissue employing a fiber optic probe with a uniformly illuminated array of collection fibers. Uniform illumination reduces local power density to avoid damage to specimens. Non-confocal operation provides efficient signal collection, and together with NIR laser excitation (785 nm diode laser) allows good depth penetration enabling recovery of spectra from beneath the skin. Multivariate data reduction is used to resolve Raman spectra of bone tissue from the spectra generated from overlying tissue. The probe utilizes non-confocal optics and uniform illumination allowing the system to collect spectra from above and below the range of best focus while applying a low power density. Despite extensive photon migration in the tissue specimens, the system can resolve transcutaneous signals because the collection cone of each fiber is asymmetric with respect to the center of illumination. Here we report preliminary results of tissue specimens taken from chicken tibia as well as from a human elbow.

We have designed and constructed a multimodal multiplex Raman spectrometer which uses multi-wavelength excitation to better detect signals in the presence of fluorescence by taking advantage of the shift-variance of the Raman signal with respect to excitation frequency. Coupled with partial-least-squares (PLS) regression, the technique applied to ethanol estimation in a tissue phantom achieves root-mean-squared-cross-validation errors (RMSCVE) of 9.2 mmol/L with a model formed with 2 principal components, compared to a single wavelength data set with equivalent energy where 7 principal components were used to achieve an RMSCVE of 39.1 mmol/L.

The purpose of this study was to explore the feasibility of using near-infrared (NIR) Raman spectroscopy and multivariate techniques for distinguishing cancer from normal and benign tissue in the colon. A total of 105 colonic specimens were used for Raman studies including 41 normal, 18 polyps, and 46 malignant tumors. The multivariate statistical techniques such as PCA-SVM were utilized to extract the significant Raman features and to develop effective diagnostic algorithms for tissue classification. The results showed that high-quality Raman spectra in the 800-1800 cm^-1 range can be acquired from human colonic tissues in vitro, and Raman spectra differed significantly between normal, benign and malignant tumor tissue. PCA-SVM yielded a diagnostic sensitivity of 100%, 100%, and 97.7%, and specificity of 99.8%, 100%, and 100%, respectively, for differentiation between normal, polyp, and malignant tissue. Therefore, NIR Raman spectroscopy associated with multivariate techniques provides a significant potential for the noninvasive diagnosis of colonic cancers in vivo based on optical evaluation of biomolecules.

Electron multiplying CCDs have revolutionized the world of low light imaging by bringing improved detection limits along with high readout rates; however, it remains to be seen whether they offer any benefits to the world of spectroscopy. Here we review the performance of current CCD and EMCCD detectors and compare their performance for low light level spectroscopy applications. In particular, we look at the detection limits of both technologies for real applications and examine all the parameters that affect these limits in a practical situation. We compare sensitivities, dark signal, noise factors and readout noise with the latter two as the ultimate limitations for detection. We also look at emerging new technology for low light spectroscopy applications which optimizes the parameters discussed without the disadvantages of the current technologies. Theoretical signal to noise data comparing conventional CCD and EMCCD technologies is presented and discussed. Experimental signal to noise comparisons are made for Raman spectra obtained using both conventional and electron multiplying CCDs in conjunction with a confocal Raman microscope. It is concluded that traditional CCDs have a superior detection limit and equal sensitivity to that of EMCCDs and are far superior for high quality quantitative data measurements.

The biochemical composition of mammalian cells has been estimated by Raman spectroscopy and the results compared with other biochemical methods. The Raman spectroscopy estimates were performed by fitting measured Raman and infrared spectra of dense cell suspensions to a linear combination of basis components (RNA, DNA, protein, lipid, glycoen). The Raman spectroscopy results are compared to biochemical analyses performed by extraction and quantfication of the biochemical components. Both absolute and relative measurements of biochemical composition are compared. Both the Raman and biochemical results indicate that there are signficant differences in gross biochemical composition dependent on growth stage and tumorigneicity.

Advances in technologies have brought us closer to routine spectroscopic diagnosis of early malignant disease. However, there is still a poor understanding of the carcinogenesis process. For example it is not known whether many cancers follow a logical sequence from dysplasia, to carcinoma in situ, to invasion. Biochemical tissue changes, triggered by genetic mutations, precede morphological and structural changes. These can be probed using Raman or FTIR microspectroscopy and the spectra analysed for biochemical constituents. Local microscopic distribution of various constituents can then be visualised. Raman mapping has been performed on a number of tissues including oesophagus, breast, bladder and prostate. The biochemical constituents have been calculated at each point using basis spectra and least squares analysis. The residual of the least squares fit indicates any unfit spectral components. The biochemical distribution will be compared with the defined histopathological boundaries. The distribution of nucleic acids, glycogen, actin, collagen I, III, IV, lipids and others appear to follow expected patterns.

Necrosis is the dominant form of cell-death that results from several modalities of cancer treatment. An estimate of post-treatment necrosis serves as an useful indicator of treatment efficacy and tumor response. A non-invasive means of identifying necrosis would serve as a useful clinical tool. In this study, we use Raman spectroscopy for the biochemical characterization of necrosis. Necrosis formation in tissue has been modeled in vitro by the use of multicellular spheroids. The relative amounts of various biochemical components have been estimated and correlated with quantitative estimates of necrosis.

Breast calcifications can be found in both benign and malignant lesions and the composition of these calcifications can indicate the possible disease state. Calcium oxalate dihydrate (COD) is found to be associated with benign lesions, however calcium hydroxyapatite (HAP) is found mainly in malignant tissue. As current practices such as mammography and histopathology examine the morphology of the specimen, they can not reliably distinguish between the two types of calcification, which frequently are the only features that indicate the presence of a cancerous lesion. Therefore this information can be used to make a simplistic diagnostic decision, if the biochemistry of the calcifications can be probed. Studies have been performed utilising the synchrotron mid-IR beamline at Daresbury (UK) to probe the local tissue biochemistry around breast calcifications. Raman and FTIR spectroscopic analysis of the same specimen have also been performed and spectral maps have been collected of areas in and around calcifications. Principal component analysis was used to identify the major differences in the spectra across each map. FTIR and Raman spectroscopic techniques provide complementary biochemical information and demonstrate great potential for determining biochemical changes in calcified breast tissue. Further studies will be carried out using these techniques to investigate the formation mechanisms and effects of hydroxyapatite on breast tissue and to correlate the type of hydroxyapatite present to the tumour grade.

In this work we employ the Fourier Transform Raman Spectroscopy to study the human breast tissues, both normal and pathological. In the present study we analyze 194 Raman spectra from breast tissues that were separated into 9 groups according to their corresponding histopathological diagnosis, which are as follows: Normal breast tissue, Fibrocystic condition, In Situ Duct Carcinoma, In Situ Duct Carcinoma with Necrosis, Infiltrating Duct Carcinoma, Infiltrating Duct Inflammatory Carcinoma, Infiltrating Duct Medullar Carcinoma, Infiltrating Duct Colloid Carcinoma, and Infiltrating Lobule Carcinoma. We found a strong lipids Raman band, and this structure was identified as abundant in the normal breast tissue spectra. The primary structure of proteins was identified through the shift of the amine acids bands. The identification of the secondary structure of proteins occurred through the peptide bands (Amide I and Amide III). In relation to the carbohydrates, the spectra of duct infiltrating colloid carcinoma, fibrocystic condition, and infiltrating duct carcinoma have been compared and identified. We observed an increase in the intensity of the 800-1200 cm^-1 spectral region. This fact could indicate the presence of liquid cystic. We also notice alterations in the peaks in the region of 500 to 600 cm^-1 and 2000 to 2100 cm^-1 that may suggest changes in the nucleic acids of the cells.

Tip-enhanced Raman scattering (TERS), a scattering type scanning near-field optical microscope (s-SNOM), is a technique which combines Raman spectroscopy with an optically active silver coated scanning probe microscopy tip. Only the molecules located in a small area close to the tip apex will experience the field enhancement. This provides a unique tool to obtain highly enhanced Raman signals together with a high lateral-resolution. In this paper, preliminary investigation of the interaction between methylene blue and calf-thymus double-stranded (ds) DNA has been carried out by SERS. Furthermore, TERS spectra of methylene blue have been collected for the first time, and are compared with standard surface enhanced Raman scattering (SERS). The results are discussed in terms of possible biological applications.

Near infrared spectroscopy exhibits a tremendous potential for clinical chemistry and tissue pathology. Owing to its penetration depth into human skin, near infrared radiation can probe chemical and structural information non-invasively. Metabolic diseases such as diabetes mellitus increase nonenzymatic glycation with the effect of glucose molecules bonding chemically to proteins. In addition, glycation accumulates on tissue proteins with the clearest evidence found in extracellular skin collagen, affecting also covalent crosslinking between adjacent protein strands, which reduces their flexibility, elasticity, and functionality. Non-enzymatically glycated proteins in human skin and following chemical and structural skin changes were our spectroscopic target. We carried out measurements on 109 subjects using two different NIR-spectrometers equipped with diffuse reflection accessories. Spectra of different skin regions (finger and hand/forearm skin) were recorded for comparison with clinical blood analysis data and further patient information allowing classification into diabetics and non-diabetics. Multivariate analysis techniques for supervised classification such as linear discriminant analysis (LDA) were applied using broad spectral interval data or a number of optimally selected wavelengths. Based on fingertip skin spectra recorded by fiber-optics, it was possible to classify diabetics and non-diabetics with a maximum accuracy of 87.8 % using leave-5-out cross-validation (sensitivity of 87.5. %, specificity of 88.2 %). With the results of this study, it can be concluded that ageing and glycation at elevated levels cannot always be separated from each other.

In the last few years, Raman spectroscopy has been increasingly used for the characterization of normal and pathological tissues. A new Raman system, constituted of optic fibers bundle coupled to an axial Raman spectrometer (Horiba Jobin Yvon SAS), was developed for in vivo investigations. Here, we present in vivo analysis on two tissues: human skin and esophagus mucosa on a rat model. The skin is a directly accessible organ, representing a high diversity of lesions and cancers. Including malignant melanoma, basal cell carcinoma and the squamous cell carcinoma, skin cancer is the cancer with the highest incidence worldwide. Several Raman investigations were performed to discriminate and classify different types of skin lesions, on thin sections of biopsies. Here, we try to characterize in vivo the different types of skin cancers in order to be able to detect them in their early stages of development and to define precisely the exeresis limits. Barrett's mucosa was also studied by in vivo examination of rat's esophagus. Barrett's mucosa, induced by gastro-esophageal reflux, is a pretumoral state that has to be carefully monitored due to its high risk of evolution in adenocarcinoma. A better knowledge of the histological transformation of esophagus epithelium in a Barrett's type will lead to a more efficient detection of the pathology for its early diagnosis. To study these changes, an animal model (rats developing Barrett's mucosa after duodenum - esophagus anastomosis) was used. Potential of vibrational spectroscopy for Barrett's mucosa identification is assessed on this model.

Background fluorescence can often complicate the use of Raman microspectroscopy in the study of musculoskeletal tissues. Such fluorescence interferences are undesirable as the Raman spectra of matrix and mineral phases can be used to differentiate between normal and pathological or microdamaged bone. Photobleaching with the excitation laser provides a non-invasive method for reducing background fluorescence, enabling 532 nm Raman hyperspectral imaging of bone tissue. The signal acquisition time for a 400 point Raman line image is reduced to 1-4 seconds using electronmultiplying CCD (EMCCD) detector, enabling acquisition of Raman images in less than 10 minutes. Rapid photobleaching depends upon multiple scattering effects in the tissue specimen and is applicable to some, but not all experimental situations.