This PDF file contains the front matter associated with SPIE Proceedings Volume 8087, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

A rapid label-free approach for molecular histopathology is presented and reviewed. Broadband vibrational spectra are generated by nonlinear interferometric vibrational imaging (NIVI), a coherent anti-Stokes Raman scattering (CARS)- based technique that uses interferometry and signal processing approaches to acquire Raman-like profiles with suppression of the non-resonant background. This allows for the generation of images that provide contrast based on quantitative chemical composition with high spatial and spectral resolution. Algorithms are demonstrated for reducing the diagnostic spectral information into color-coded composite images for the rapid identification of chemical constituents in skin, as well as differentiating normal from abnormal tissue in a pre-clinical tumor model for human breast cancer. This technology and methodology could result in an alternative method to the traditional histological staining and subjective interpretation procedure currently used in the diagnosis of disease, and has the potential for future in vivo molecular histopathology.

Support Vector Machines have been used successfully for the classification of data in a wide range of applications. A key factor affecting the accuracy of the classification is the choice of kernel. In this paper we propose the use of Support Vector Machines with a correlation kernel. The correlation kernel is an appropriate choice when performing classification of Raman spectra because it reduces the need for pre-processing. Pre-processing can greatly affect the accuracy of the results because it introduces user bias and over-fitting effects. The correlation kernel is "self-normalizing" and produces superior classification performance with minimal pre-processing. Our results show that the performance on highly-noisy data, obtained using inexpensive equipment, is still high even when the classification is applied on a distinct hold-out set of test data. This is an important consideration when developing clinically viable diagnostic applications.

This contribution will present a variety of applications of lab-on-a-chip surface enhanced Raman spectroscopy in the field of bioanalytic. Beside the quantification and online monitoring of drugs and pharmaceuticals, determination of enzyme activity and discrimination of bacteria are successfully carried out utilizing LOC-SERS. The online-monitoring of drugs using SERS in a microfluidic device is demonstrated for nicotine. The enzyme activity of thiopurine methyltransferase (TPMT) in lysed red blood cells is determined by SERS in a lab-on-a-chip device. To analyse the activity of TPMT the metabolism of 6-mercaptopurine to 6-methylmercaptopurine is investigated. The discrimination of bacteria on strain level is carried out with different E. coli strains. For the investigations, the bacteria are busted by ultra sonic to achieve a high information output. This sample preparation provides the possibility to detect SERS spectra containing information of the bacterial cell walls as well as of the cytoplasm. This contribution demonstrates the great potential of LOC-SERS in the field of bioanalytics.

Pathogen detection is essential without time delay especially for severe diseases like sepsis. Here, the survival rate is dependent on a prompt antibiosis. For sepsis three hours after the onset of shock the survival rate of the patient drops below 60 %. Unfortunately, the results from standard diagnosis methods like PCR or microbiology can normally be received after 12 or 36 h, respectively. Therefore diagnosis methods which require less cultivation or even no cultivation at all have to be established for medical diagnosis. Here, Raman spectroscopy, as a vibrational spectroscopic method, is a very sensitive and selective approach and monitors the biochemical composition of the investigated sample. Applying micro-Raman spectroscopy allows for a spatial resolution below 1 μm and is therefore in the size range of bacteria. Raman spectra of bacteria depend on the physiological status. Therefore, the databases require the inclusion of the necessary environmental parameters such as temperature, pH, nutrition, etc. Such large databases therefore require a specialized chemometric approach, since the variation between different strains is small. In this contribution we will demonstrate the capability of Raman spectroscopy to identify pathogens without cultivation even from real environmental or medical samples.

The objective of this study was to demonstrate the utility of optical cryoimaging and fluorometry to evaluate tissue redox state of the mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (Flavin Adenine Dinucleotide) in intact rat lungs. The ratio (NADH/FAD), referred to as mitochondrial redox ratio (RR), is a measure of the lung tissue mitochondrial redox state. Isolated rat lungs were connected to a ventilation-perfused system. Surface NADH and FAD fluorescence signals were acquired before and after lung perfusion in the absence (control perfusate) or presence of potassium cyanide (KCN, complex IV inhibitor) to reduce the mitochondrial respiratory chain (state 5 respiration). Another group of lungs were perfused with control perfusate or KCN-containing perfusate as above, after which the lungs were deflated and frozen rapidly for subsequent 3D cryoimaging. Results demonstrate that lung treatment with KCN increased lung surface NADH signal by 22%, decreased FAD signal by 8%, and as result increased RR by 31% as compared to control perfusate (baseline) values. Cryoimaging results also show that KCN increased mean lung tissue NADH signal by 37%, decreased mean FAD signal by 4%, and increased mean RR by 47%. These results demonstrate the utility of these optical techniques to evaluate the effect of pulmonary oxidative stress on tissue mitochondrial redox state in intact lungs.

In many fields such as biomedical or food industry, surface colonization by micro-organisms leads to biofilms formation that are tridimentional biostructures highly resistant to the action of antimicrobials, by mechanisms still unclear. In order to deepen our understanding of the initial interaction of bacteria cells with a solid surface, we analyze by in situ vibrational Sum Frequency Generation (SFG) spectroscopy the effect of the adhesion of hydrophilic Lactoccocus lactis bacteria and its hydrophobic mutants in distilled water on a self-assembled monolayer (SAM) of octadecanethiol (ODT) on a gold film. When a homogeneous bacterial monolayer is deposited on this ordered surface, SFG spectrum of the ODT SAM shows significant intensity changes from that in air or in water. Its modelling as a function of conformation allows to distinguish optical effects due to the water solution surrounding bacteria from conformational changes of the ODT SAM due to the presence of the bacteria cells. Futhermore, bacterial adhesion induces different measurable effects on the ODT SAM conformation, depending on the hydrophobic / hydrophilic character of the bacterial surface. Such a result deserves to be taken into account for the design of new materials with improved properties or to control biofilm formation.

In this work, we report advances in the fabrication and anticipated performance of a polymer biosensor photonic chip developed in the European Union project P3SENS (FP7-ICT4-248304). Due to the low cost requirements of point-ofcare applications, the photonic chip is fabricated from nanocomposite polymeric materials, using highly scalable nanoimprint- lithography (NIL). A suitable microfluidic structure transporting the analyte solutions to the sensor area is also fabricated in polymer and adequately bonded to the photonic chip. We first discuss the design and the simulated performance of a high-Q resonant cavity photonic crystal sensor made of a high refractive index polyimide core waveguide on a low index polymer cladding. We then report the advances in doped and undoped polymer thin film processing and characterization for fabricating the photonic sensor chip. Finally the development of the microfluidic chip is presented in details, including the characterisation of the fluidic behaviour, the technological and material aspects of the 3D polymer structuring and the stable adhesion strategies for bonding the fluidic and the photonic chips, with regards to the constraints imposed by the bioreceptors supposedly already present on the sensors.

The current gold standard method of Malaria diagnosis relies on the blood smears examination. The method is laborintensive, time consuming and requires the expertise for data interpretation. In contrast, Raman scattering from a metabolic byproduct of the malaria parasite (Hemozoin) shows the possibility of rapid and objective diagnosis of malaria. However, hemozoin concentration is usually extremely low especially at the early stage of malaria infection, rendering weak Raman signal. In this work, we propose the sensitive detection of enriched β-hematin, whose spectroscopic properties are equivalent to hemozoin, based on surface enhanced Raman spectroscopy (SERS) by using magnetic nanoparticles. A few orders of magnitude enhancement in the Raman signal of β-hematin can be achieved using magnetic nanoparticles. Furthermore, the effect of magnetic field on SERS enhancement is investigated. Our result demonstrates the potential of SERS using magnetic nanoparticles in the effective detection of hemozoin for malaria diagnosis.

Due to its high lateral resolution, Raman microspectrsocopy is rapidly becoming an accepted technique for the subcellular imaging of single cells. Although the potential of the technique has frequently been demonstrated, many improvements have still to be realised to enhance the relevancy of the data collected. Although often employed, chemical fixation of cells can cause modifications to the molecular composition and therefore influence the observations made. However, the weak contribution of water to Raman spectra offers the potential to study live cells cultured in vitro using an immersion lens, giving the possibility to record highly specific spectra from cells in their original state. Unfortunately, in common 2-D culture models, the contribution of the substrates to the spectra recorded requires significant data pre-processing causing difficulties in developing automated methods for the correction of the spectra. Moreover, the 2-D in vitro cell model is not ideal and dissimilarities between different optical substrates within in vitro cell cultures results in morphological and functional changes to the cells. The interaction between the cells and their microenvironment is crucial to their behavior but also their response to different external agents such as radiation or anticancer drugs. In order to create an experimental model closer to the real conditions encountered by the cell in vivo, 3-D collagen gels have been evaluated as a substrate for the spectroscopic study of live cells. It is demonstrated that neither the medium used for cell culture nor the collagen gels themselves contribute to the spectra collected. Thus the background contributions are reduced to that of the water. Spectral measurements can be made in full cell culture medium, allowing prolonged measurement times. Optimizations made in the use of collagen gels for live cells analysis by Raman spectroscopy are encouraging and studying live cells within a collagenous microenvironment seems perfectly accessible.

Circulating epithelial tumor cells are of increasing importance for tumor diagnosis and therapy monitoring of cancer patients. The definite identification of the rare tumor cells within numerous blood cells is challenging. Therefore, within the research initiative "Jenaer Zell-Identifizierungs-Gruppe" (JenZIG) we develop new methods for cell identification, micromanipulation and sorting based on spectroscopic methods and microfluidic systems. In this contribution we show, that classification models based on Raman spectroscopic analysis allow a precise discrimination of tumor cells from non-tumor cells with high prediction accuracies, up to more than 99% for dried cells. That holds true for unknown cell mixtures (tumor cells and leukocytes/erythrocytes) under dried conditions as well as in solution using the Raman laser as an optical tweezers to keep the cells in focus. We extended our studies by using a capillary system consisting of a quartz capillary, fiber optics and an adjustable fitting to trap cells. This system allows a prediction accuracy of 92.2% on the single cell level, and is a prerequisite for the development of a cell sorting and identification device based on a microfluidic chip. Initial experiments show that tumor cell lines can be differentiated from healthy leukocyte cells with an accuracy of more than 98%.

Worldwide, yearly about 450,000 women die from the consequences of breast cancer. Current imaging modalities are not optimal in discriminating benign from malignant tissue. Visualizing the malignancy-associated increased hemoglobin concentration might significantly improve early diagnosis of breast cancer. Since photoacoustic imaging can visualize hemoglobin in tissue with optical contrast and ultrasound-like resolution, it is potentially an ideal method for early breast cancer imaging. The Twente Photoacoustic Mammoscope (PAM) has been developed specifically for breast imaging. Recently, a large clinical study has been started in the Medisch Spectrum Twente in Oldenzaal using PAM. In PAM, the breast is slightly compressed between a window for laser light illumination and a flat array ultrasound detector. The measurements are performed using a Q-switched Nd:YAG laser, pulsed at 1064 nm and a 1 MHz unfocused ultrasound detector array. Three-dimensional data are reconstructed using a delay and sum reconstruction algorithm. Those reconstructed images are compared with conventional imaging and histopathology. In the first phase of the study 12 patients with a malignant lesion and 2 patients with a benign cyst have been measured. The results are used to guide developments in photoacoustic mammography in order to pave the way towards an optimal technique for early diagnosis of breast cancer.

Conventional white light endoscopy (WLE) is the most widespread technique used today for colorectal cancer diagnosis and is considered as the gold standard when coupled to biopsy and histology. However for early stage colorectal cancer diagnosis, which is very often characterised by flat adenomas, the use of WLE is quite difficult due to subtle or quasiinvisible morphological changes of the colonic lining. Figures worldwide point out that diagnosing colorectal cancer in its early stages would significantly reduce the death toll all while increasing the 5-year survival rate. Several techniques are currently being investigated in the scope of providing new tools that would allow such a diagnostic or assist actual techniques in so doing. We hereby present a novel technique where High spatial Resolution MRI (HR-MRI) is coupled to optical spectroscopy (autofluorescence and reflectance) in a bimodal endoluminal probe to extract morphological data and biochemical information respectively. The design and conception of the endoluminal probe along with the preliminary results obtained with an organic phantom and in-vivo (rabbit) are presented and discussed.

Histopathology provides the gold standard assessment of colonoscopic biopsies. Infrared spectroscopy can potentially map biochemical changes across a tissue section identifying disease. The purpose of this study was to determine if infrared spectroscopy could classify different colorectal pathologies and to investigate biochemical composition. Colonoscopic tissue biopsies were snap frozen at colonoscopy. 10 micron thick sections were mounted on CaF₂ slides. 3- D spectral datasets (2 spatial dimensions and one spectral) were measured from thawed specimens using a Perkin Elmer infrared imaging system in transmission mode. Contiguous tissue sections stained with H&E were reviewed by a specialist gastrointestinal pathologist for comparison. Tissue spectra from epithelial tissues were classified using principal components fed linear discriminant analysis with leave one out cross validation. Reference spectra from purchased biochemicals (Sigma-Aldrich) were measured. Ordinary least squares analysis estimated the relative biochemical signal contribution from epithelial regions. Spectra from tissue epithelia measured from normal tissue, hyperplastic polyps, adenomatous polyps, cancer and ulcerative colitis samples were classified with accuracies in excess of 90%. Ordinary least squares analysis demonstrated a higher DNA to cytoplasm ratio in cancer compared to normal tissue. FTIR spectra from epithelia can be used to classify colorectal pathologies with high accuracy. Ordinary least squares analysis shows promise for extraction of useful biochemical information. These techniques could aid the histopathologist and ultimately lead to automated histopathological processing.

We report novel bioconjugated nanosensitizers as optical and therapeutic probes for the detection, monitoring and treatment of cancer. These nanosensitisers, consisting of hypericin loaded bioconjugated gold nanoparticles, can act as tumor cell specific therapeutic photosensitizers for photodynamic therapy coupled with additional photothermal effects rendered by plasmonic heating effects of gold nanoparticles. In addition to the therapeutic effects, the nanosensitizer can be developed as optical probes for state-of-the-art multi-modality in-vivo optical imaging technology such as in-vivo 3D confocal fluorescence endomicroscopic imaging, optical coherence tomography (OCT) with improved optical contrast using nano-gold and Surface Enhanced Raman Scattering (SERS) based imaging and bio-sensing. These techniques can be used in tandem or independently as in-vivo optical biopsy techniques to specifically detect and monitor specific cancer cells in-vivo. Such novel nanosensitizer based optical biopsy imaging technique has the potential to provide an alternative to tissue biopsy and will enable clinicians to make real-time diagnosis, determine surgical margins during operative procedures and perform targeted treatment of cancers.

Vibrational spectroscopic imaging methods are novel tools to visualise chemical component in tissue without staining. Fourier transform infrared (FTIR) imaging is more frequently applied than Raman imaging so far. FTIR images recorded with a FPA detector have been demonstrated to identify the primary tumours of brain metastases. However, the strong absorption of water makes it difficult to transfer the results to non-dried tissues. Raman spectroscopy with near infrared excitation can be used instead and allows collecting the chemical fingerprint of native specimens. Therefore, Raman spectroscopy is a promising tool for tumour diagnosis in neurosurgery. Scope of the study is to compare FTIR and Raman images to visualize the tumour border and identify spectral features for classification. Brain metastases were obtained from patients undergoing surgery at the university hospital. Brain tissue sections were shock frozen, cryosectioned, dried and the same areas were imaged with both spectroscopic method. To visualise the chemical components, multivariate statistical algorithms were applied for data analysis. Furthermore classification models were trained using supervised algorithms to predict the primary tumor of brain metastases. Principal component regression (PCR) was used for prediction based on FTIR images. Support vector machines (SVM) were used for prediction based on Raman images. The principles are shown for two specimens. In the future, the study will be extended to larger data sets.

A specialized transient digitizer system was developed for spectroscopic collection of fluorescence wavelength-time matrices (WTMs) from biological tissues. The system is compact, utilizes fiber optic probes for clinical compatibility, and offers rapid collection of high signal-to-noise ratio (>100) time- and wavelength- resolved fluorescence. The system is compatible with excitation sources operating in excess of 25 kHz. Wavelength-resolved measurement range is 300-800 nm with ≥ 0.01 nm steps. Time-resolved measurement depth is 128 ns with fixed 0.2 ns steps. The information-rich WTM data provides comprehensive fluorescence sensing capabilities, as demonstrated on tissue simulating phantoms. Extracting wavelength-resolved fluorophore lifetimes illustrates the potential of using the technology to resolve exogenous or endogenous fluorophore contributions in tissue samples in a clinical setting for tissue diagnostics and monitoring.

Collagen is the major component of skin, tendon, cartilage, cornea, and, as a main structural protein it is the key determinant of thermo-mechanical properties of collagen-rich tissues in mammals. Thermal damage of chicken dermis and tendon, bovine leg tendon, and other collagen contained tissues were investigated with the use of second harmonic generation (SHG) and two-photon excited auto-fluorescence microscopy and spectroscopy. Samples were heating in a temperature-controlled water bath in the temperature range 18-90° C. SHG time-lapse imaging and analysis of intensity decay showed that the collagen thermal destruction depended on both temperature and heating time, and can be modeled by the Arrhenius equation. Temporal decay of SHG signal from the chicken dermis was single exponential during isothermal treatment at temperatures above 60º C that allowed to determine activation energy and frequency factor of skin collagen denaturation. Furthermore, two-exponential decay and partially reversible change in SHG intensity were registered during the tendon thermal treatment. A simple laser system and procedure is proposed for a real-time monitoring of collagen fiber thermal modification within a microscopic volume of 1 nl.

This paper presents a hyper-spectral video endoscopy system which utilizes a combination of auto-fluorescence imaging and white-light reflectance spectroscopy for intra-surgery tissue classification. The results of the first clinical study consisting of 59 cases of otolaryngoscopic examinations and thorax surgeries are discussed in this paper. The main focus of this application is the detection of tumor tissue, although hyper-spectral video endoscopy is not limited to cancer detection. The results show that hyper-spectral video endoscopy exhibits a large potential to become an important imaging technology for medical imaging devices that provide additional diagnostic information about the tissue under investigation.

Collagen makes up one third of the total protein in humans, being formed by the connection of three polypeptide chains arranged in a triple helix. This protein has fundamental importance in the formation of extracellular matrix of connective tissue. This study aimed to analyze the structural changes of collagen, which are resulting from inflammatory processes in oral mucosa, and to make the comparative analysis between the histopathology and the Raman spectra. The samples of tissues with inflammatory fibrous hyperplasia (IFH) and normal mucosa (NM) were evaluated by Raman Spectroscopy, hematoxylin-eosin and Massons trichrome stain. The histological analysis in both stains showed differences in collagen fibers, which was presented as thin fibers and arranged in parallel direction in NM and as collagen fibers are thick, mature and not organized, showing that these types of stain show morphological changes of collagen in IFH. The Raman Spectroscopy discriminate the groups of NM and IFH based on vibrational modes of proline, hydroxiproline and CH₃, CH₂. The histological stains only shows information from morphological data, and can be complemented by Raman spectra. This technique could demonstrate that inflammatory process caused some changes in collagen structure which is related to aminoacids such as proline and hidroxyproline.

Pancreatic adenocarcinoma has a five-year survival rate of only 6%, largely because current diagnostic methods cannot reliably detect the disease in its early stages. Reflectance and fluorescence spectroscopies have the potential to provide quantitative, minimally-invasive means of distinguishing pancreatic adenocarcinoma from normal pancreatic tissue and chronic pancreatitis. The first collection of wavelength-resolved reflectance and fluorescence spectra and time-resolved fluorescence decay curves from human pancreatic tissues was acquired with clinically-compatible instrumentation. Mathematical models of reflectance and fluorescence extracted parameters related to tissue morphology and biochemistry that were statistically significant for distinguishing between pancreatic tissue types. These results suggest that optical spectroscopy has the potential to detect pancreatic disease in a clinical setting.

The nude mice injected with human gastric cancer cells (SGC-7901) in their peritoneums were chosen as the animal models of gastric cancer peritoneal dissemination in this research. The Raman spectra at 785nm excitation of both these nude mice which were in different tumor planting periods and the normal counterpart were taken in vivo in the imitate laparotomy. 205 spectra were collected. The spectra of different tissue types were compared and classified by Support Vector Machine (SVM) algorithm. Significant differences were showed between normal and malignant tissues. The gastric cancer nodules had lower Raman intensities at 870, 1330, 1450, and 1660cm^-1, but higher at 1007, 1050, 1093 and 1209cm^-1, compared with normal tissues. Additionally, the spectra of malignant tissues had two peaks around 1330 cm^-1 (1297cm^-1 and 1331cm^-1), while the spectra of normal tissues had only one peak (1297cm^-1). The differences were attributed to the intensities of the stretching bands of the nucleic acid, protein and water. These features could be used to diagnose gastric cancer. The Support Vector Machine (SVM) algorithm was used to classify these spectra. For normal and malignant tissues, the sensitivity, specificity and accuracy were 95.73%, 70.73% and 90.73%, respectively, while for different tumor planting periods, they were 98.82%, 98.73% and 98.78%. The experimental results show that Raman spectra differ significantly between cancerous and normal gastric tissues, which provides the experimental basis for the diagnosis of gastric cancer by Raman spectroscopy technology. And SVM algorithm can give the well generalized classification performance for the samples, which expands the application of mathematical algorithms in the classification.

Chromophore concentrations from skin contain information about the blood parameters, for example total hemoglobin content or antioxidant status of the skin. Deviations from the normal values of the concentrations may indicate pathologies. As the chromophore concentrations are determined from skin absorption coefficients, the optical absorption spectra of the isolated skin chromophores have to be known in advance, enabling least squares fitting of the basis spectra to the skin absorption coefficient. It could be shown that spectrally and spatially resolved reflectance in combination with a determination of absorption and reduced scattering coefficients from a look-up table provides a means for quantification of chromophores, although the accuracy largely depends on the tissue model. Good qualitative results can also be obtained with the homogenous tissue model used here. For example, it could be shown that the hemoglobin basis spectra determined from human whole blood and the pure water absorption fit very well to the skin absorption coefficients, but the ex vivo carotene spectra does not. Therefore it was examined how the carotene spectra change from ex vivo to in vivo. Interindividual and interpositional variation of the optical parameters could also be evaluated using this method as well as the dependence of determined optical parameters on the source-detector separation.

Hyperspectral fluorescence imaging is a modality combining high spatial and spectral resolution with increased sensitivity for low photon counts. The main objective of the current study was to investigate if this technique is a suitable tool for characterization of diffusion properties in human skin. This was done by imaging fluorescence from Alexa 488 in ex vivo human skin samples using an sCMOS based hyperspectral camera. Pre-treatment with acetone, DMSO and mechanical micro-needling of the stratum corneum created variation in epidermal permeability between the measured samples. Selected samples were also stained using fluorescence labelled biopolymers. The effect of fluorescence enhancers on transdermal diffusion could be documented from the collected data. Acetone was found to have an enhancing effect on the transport, and the results indicate that the biopolymers might have a similar effect, The enhancement from these compounds were not as prominent as the effect of mechanical penetration of the sample using a micro-needling device. Hyperspectral fluorescence imaging has thus been proven to be an interesting tool for characterization of fluorophore diffusion in ex vivo skin samples. Further work will include repetition of the measurements in a shorter time scale and mathematical modeling of the diffusion process to determine the diffusivity in skin for the compounds in question.

Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH. This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.

Psoriasis is an autoimmune disease of the skin characterized by hyperkeratosis, hyperproliferation of the epidermis, inflammatory cell accumulation and increased dilatation of dermal papillary blood vessels. Cases of psoriasis were investigated in vivo with optical means in order to evaluate the potential of in vivo optical biopsy. A Polarization Multispectral Dermoscope was employed for the macroscopic observation. Features such as the 'dotted' blood vessels pattern was observed with high contrast. High resolution image sections of the epidermis and the dermis were produced with a custom made Multiphoton Microscope. Imaging extended from the surface of the lesion down to the papillary dermis, at a depth of 200 μm. In the epidermis, a characteristic morphology of the stratum corneum found only in Psoriasis was revealed. Additionally, the cytoplasmic area of the cells in the stratum spinosum layer was found to be smaller than normal. In the dermis the morphological features were more pronounced, where the elongated dermal papillae dominated the papillary layer. Their length exceeds 100μm, which is a far greater value compared to that of healthy skin. These in vivo observations are consistent with the ex vivo histopathological observations, supporting both the applicability and potentiality of multispectral dermoscopy and multiphoton microscopy in the field of in vivo optical investigation and biopsy of skin.

Second-harmonic generation and two-photon excited fluorescence microscopy were used in combination in the same optical system for in vivo imaging. This work aimed at detecting collagen remodeling and reorganization in living subjects following laser micro-ablative fractional resurfacing treatment. Treated regions in the forearm of volunteers covering a wide age range were imaged with two-photon microscopy before and forty days after the treatment. A strong age-dependence of the treatment effectiveness was found, demonstrating a negligible effect in very young subjects (age < 35 years) face to a mild effect on mid-age subjects (35 years < age < 60 years) and a significant synthesis of new collagen in the most aged subjects (age > 60 years). The amount of newly synthesized collagen as well as its organization were evaluated by means of both visual examination of two-photon images and an image analysis methods, based on second-harmonic to autofluorescence ageing index of dermis (SAAID) scoring. The obtained results demonstrate the performance of laser fractional micro ablative resurfacing without the need for an invasive biopsy.

Atherosclerotic plaques are mainly composed of proteoglycans, triglycerides, cholesterol, cholesterolester and crystalline calcium. From histopathological characterizations it is known that the composition of these atherosclerotic plaques can vary to a great extent, due to different risk factors as smoking, hyperlipedemia, or genetic background ect. The individual plaque components can be spectroscopically easily identified. Furthermore, spectroscopic imaging technologies offer the possibility to study the plaque compositions in a more quantitative manner than traditional staining techniques. Here, we compare the potential of IR, Raman and CARS microscopy to characterize the constitution of atherosclerotic plaques as well as the structure of the surrounding tissue. For data analysis and image reconstruction spectral decomposition algorithms such as vertex component analysis (VCA) were introduced. The results are in good agreement with the histopathology. Aim of the study is to correlate the compositional characteristics of atherosclerotic plaques with individual disease patterns.

Retinal diseases like age-related macular degeneration have become an important cause of visual loss depending on increasing life expectancy and lifestyle habits. Due to the fact that no satisfying treatment exists, early diagnosis and prevention are the only possibilities to stop the degeneration. The protein cytochrome c (cyt c) is a suitable marker for degeneration processes and apoptosis because it is a part of the respiratory chain and involved in the apoptotic pathway. The determination of the local distribution and oxidative state of cyt c in living cells allows the characterization of cell degeneration processes. Since cyt c exhibits characteristic absorption bands between 400 and 650 nm wavelength, uv/vis in situ spectroscopic imaging was used for its characterization in retinal ganglion cells. The large amount of data, consisting of spatial and spectral information, was processed by multivariate data analysis. The challenge consists in the identification of the molecular information of cyt c. Baseline correction, principle component analysis (PCA) and cluster analysis (CA) were performed in order to identify cyt c within the spectral dataset. The combination of PCA and CA reveals cyt c and its oxidative state. The results demonstrate that uv/vis spectroscopic imaging in conjunction with sophisticated multivariate methods is a suitable tool to characterize cyt c under in situ conditions.

Measurements of time-resolved autofluorescence (FLIM) at the human ocular fundus of diabetic patients permit the detection of early pathologic alterations before signs of diabetic retinopathy are visible. The measurements were performed by the Jena Fluorescence Lifetime Laser Scanner Ophthalmoscope applying time-correlated single photon counting (TCSPC) in two spectral channels (K1: 490-560 nm, K2:560-700ps). The fluorescence was excited by 70 ps pulses (FWHM) at 448 nm. The decay of fluorescence intensity was triple-exponentially approximated. The frequency of amplitudes, lifetimes, and relative contributions was compared in fields of the same size and position in healthy subjects and in diabetic patients. The most sensitive parameter was the lifetime T2 in the short-wavelength channel, which corresponds to the neuronal retina. The changes in lifetime point to a loss of free NADH and an increased contribution of protein-bound NADH in the pre-stage of diabetic retinopathy.

We show that Surface Enhanced Raman spectroscopy (SERS) combined with 2D correlation and multivariate analysis provides considerable progress in using Raman microspectroscopy for cutting edge biomedical research applications such as treatment delivering in cancer living cells, the diagnosis of retina neuroinflamed tissue and the study of elastic properties of single DNA molecules.

The diffusion capabilities of free fluorophores inside the heterogeneous three dimensional structure of Staphylococcus aureus biofilm were studied by an original image-based Fluorescence Recovery After Photobleaching method. The study was extended to BODIPY-vancomycin in order to better understand the mechanisms involved in the high tolerance of the bacteria embedded in a biofilm to the antibiotic.

Since DNA is not internalized efficiently by cells, the success of gene therapy depends on the availability of carriers to efficiently deliver genetic material into target cells. Gene delivery vectors can be broadly categorized into viral and non-viral ones. Non-viral gene delivery systems are represented by cationic lipids and polymers rely on the basics of supramolecular chemistry termed "self-assembling": at physiological pH, they are cations and spontaneously form lipoplexes (for lipids) and polyplexes (for polymers) complexing nucleic acids. In this scenario, cationic polymers are commonly used as non-viral vehicles. Their effectiveness is strongly related to key parameters including DNA binding ability and stability in different environments. Time-resolved fluorescence spectroscopy of SYBR Green I (DNA dye) was carried out to characterize cationic polymer/DNA complex (polyplex) formation dispersed in aqueous solution. Both fluorescence amplitude and lifetime proved to be very sensitive to the polymer/DNA ratio (N/P ratio, +/-).

Light absorption in tissue is generally decreased when chromophores are spatially concentrated rather than being homogeneously distributed. In tissue, this applies to hemoglobin located in blood vessels (vessel packaging). In this paper, the diffusely reflected light from 41 tissue models with discrete blood vessels with diameters ranging from 6.25 to 100 μm were simulated using the Monte Carlo technique. A reverse engineering approach was then utilized to find the model that had an optimal spectral fit to each of the simulated models. The average vessel diameter was one fitting parameter in the adaptive model. The estimated vessel diameter from the optimal fit model was compared to the known diameter from the simulated models. Two different methods to calculate the vessel packaging effect were used, one existing based on a simple analytic expression and a new method based on path length distributions. Both methods had similar performance. For the new method, the absolute RMS deviation of the estimated vessel diameter was 5.5 μm for vessel diameters ≤ 25 μm, and the relative RMS deviation was 21 % for vessel diameters > 25 μm.

This paper deals with multi-classification of skin precancerous stages based on bimodal spectroscopy combining AutoFluorescence (AF) and Diffuse Reflectance (DR) measurements. The proposed data processing method is based on Discrete Cosine Transform (DCT) to extract discriminant spectral features and on Support Vector Machine to classify. Results show that DCT gives better results for AF spectra than for DR spectra. This study shows that bimodality and monitoring spectral resolution together allow an increase in diagnostic accuracy. The choice of an adequate spectral resolution always implies an increase in diagnostic accuracy. This accuracy can get as high as 79.0% when combining different distances between collecting and exciting optical fibers.

The use of nanoparticles in biomedical applications is emerging rapidly. Recent developments have led to numerous studies of noble metal nanoparticles, down to the level of single molecule detection in living cells. The application of noble metal nanoparticles in diagnostics and treatment of early stage carcinomas is the subject of many present studies. Gold nanoparticles are particularly interesting for optical biomedical applications due to their biocompatibility and moreover, their enhanced absorption cross-sections. The latter is a result of surface plasmon resonance, which can be tuned by altering the shape of the nanoparticles enabling usage of the near infrared tissue transparent optical window. This paper presents a brief overview of the variety of shapes, size and surface chemistries of the gold nanoparticles used for cancer detection and treatment, as well as their effects in different tumour models that have recently been investigated, both in vitro and in vivo.

We report on the biofunctionalization of nanodiamond surfaces in a two step procedure: chemical modification, resulting in homogeneous and defined surface coating, with following addition of ssDNA. Carboxylation, thymidine coupling and amination methods for chemical modification of diamond surfaces for further functionalization experiments were applied. To enable the coupling process, biomolecules were also chemically modified with functional groups (-NH2). FTIR spectroscopy, fluorescence microscopy and gel electrophoresis were applied for characterizing modified ND particles and bioconjugates and for controlling the coupling success.

The combination of linear and nonlinear Raman microspectroscopy has been established to be a powerful tool for biomedical diagnostics. In this contribution we discuss our recent approaches towards CARS (coherent anti-Stokes Raman scattering) based quantification of analytes, which is generally complicated by the CARS-signal strength dependence on the square of the molecular concentration and on the interplay between a molecular-specific vibrational signal and a nonresonant contribution in the signal generation. Due to these complications the quantification of analytes presents a major challenge in CARS microscopy. Here we discuss two recently developed approaches, i.e. on the one hand a simplified setup for coherent anti-Stokes Raman scattering (CARS) microscopy, which allows for recording CARS images with 30 cm^-1 excitation bandwidth for probing Raman bands between 500 and 900 cm^-1 with minimal requirements for alignment. This experimental arrangement is based on electronic switching between CARS images recorded at different Raman resonances by combining a photonic crystal fiber (PCF) as broad-band light source and an acoustooptical programmable dispersive filter (AOPDF) as tunable wavelength filter. On the other hand, we discuss how the introduction of carbon-deuterium (C-D) bonds into drug compounds constitutes a non-invasive labeling approach that allows for higher intrinsic CARS contrast to be obtained. The quantitative detection of C-deuterated drugs by Raman microspectroscopy and CARS microscopy is examined. Concentration-dependent studies on drugs with aliphatic and aromatic C-D moieties were performed in a two-channel microfluidic chip, using the corresponding non-deuterated (C-H) isotopomers as an internal reference.

We proposed the imaging-type 2-dimensional Fourier spectroscopy that is the phase-shift interferometry between the objective lights. The proposed method can measure the 2D spectral image at the limited depth. Because of the imaging optical system, the 2D spectral images can be measured in high spatial resolution. And in the depth direction, we can get the spectral distribution only in the focal plane. In this report, we mention about the principle of the proposed wide field imaging-type 2D Fourier spectroscopy. And, we obtained the spectroscopic tomography of biological tissue of mouse's ear. In the visible region, we confirmed the difference of spectral characteristics between blood vessel region and other region. In the near infrared region (λ=900nm~1700nm), we can obtain the high-contrast blood vessel image of mouse's ear in the deeper part by InGaAs camera. Furthermore, in the middle infrared region(λ=8μ~14μm), we have successfully measured the radiation spectroscopic-imaging with wild field of view by the infrared module, such as the house plants. Additionally, we propose correction geometrical model that can convert the mechanical phase-shift value into the substantial phase difference in each oblique optical axes. We successfully verified the effectiveness of the proposed correction geometrical model and can reduce the spectral error into the error range into ±3nm using the He-Ne laser whose wavelength 632.8nm.

Topical agents inducing hyperaemisation like nonivamide or nicoboxil increase cutaneous blood flow and temperature and induce erythema. It is not proven up to now whether there is also a hyperaemisation effect in skeletal muscle. This study has the objective to determine the effects of a nonivamide / nicoboxil cream on haemodynamics in skin and calf muscle via optical spectroscopy in the visible and near-infrared with a separation of changes for skin and muscle. Left and right calves of 14 healthy subjects were treated with a nonivamide / nicoboxil cream or mock administration, and cutaneous and muscle haemoglobin were measured using a combined NIRS / VIS sensor. The topical application of the cream increased the concentration of oxygenated haemoglobin and tissue oxygen saturation significantly in skin as well as in muscle of the treated legs already after 15 minutes, with stronger and faster effects in skin. In contrast, the change in deoxygenated haemoglobin was found to be small. The kinetic of all changes varied widely between the subjects. The found haemoglobin changes might explain the beneficial effect of hyperaemisation creams for the treatment of minor injuries.

We report the design of a tissue oxygen and temperature monitor. The non-invasive, fibre based device monitors tissue haemoglobin (Hb) and oxygen saturation (SO₂) and is based on white-light reflectance spectroscopy.Visible light with wavelengths in the 500 - 650nm range is utilized. The spectroscopic algorithm takes into account the tissue scattering and melanin absorption for the calculation of tissue haemoglobin concentration and oxygen saturation. The monitor can probe superficial layers of tissue with a high spatial resolution (mm³) and a high temporal resolution (40 Hz). It provides an accurate measurement with the accuracy of SO₂ at 2 % and high reliability with less than 2 % variation of continuous SO₂ measurement over 12 hours. It can also form a modular system when used in conjunction with a laser Doppler monitor, enabling simultaneous measurements of Hb, SO₂ and blood flow. We found experimentally that the influence of the source-detector separation on the haemoglobin parameters is small. This finding is discussed by Monte Carlo simulations for the depth sensitivity profile. The influence of probe pressure and the skin pigmentation on the measurement parameters are assessed before in vivo experimental data is presented. The combination with laser Doppler flowmetry demonstrates the importance of a measurement of both the haemoglobin and the blood flow parameters for a full description of blood tissue perfusion. This is discussed in experimental data on human skin during cuff occlusion and after hyperemisation by a pharmacological cream. Strong correlation is observed between tissue oxygen (Hb and SO₂) and blood flow measurements.

Here we describe a novel approach to sialidase activity estimation. Sialidases (EC 3.2.1.18, exo-α-sialidases), also known as neuraminidases, are the group of enzymes, which hydrolyze the glycoside bound between terminal sialic acid and subsequent carbohydrate residue in glycoproteins and glycolipids. Sialic acids are the group of monosaccharides with acidic properties, since they are acetylated or glycolylated derivates of neuraminic acid. Flu and some other viruses use neuraminidase activity to infect host cells. The level of sialylation was shown to be tightly connected with tumor cell invasiveness and metastatic potential, sialylation level also determines the clearance of aged or virus-infected cells. Thus, detection of sialidase activity is of primary importance for clinical diagnostics as well as life science research. The authors developed the assay for both visualization and estimation of sialidase activity in living cells. Previously known methods for sialidase activity detection required destruction of cellular material, or were low-sensitive, or provided no information on the activity localization in certain intracellular compartment. To overcome these problems, a fluorogenic neuraminidase substrate, 4-MUNA was utilized, and the method for detection of neuraminidase activity using fluorescent microscopy was proposed, it provided a high signal level and information on cellular localization of the studied enzyme. By using this approach the increase of sialidase activity on apoptotic cells was demonstrated in comparison to viable and primary necrotic cells.

In this paper, Raman spectra of human serum were measured using Raman spectroscopy, then the spectra was analyzed by multivariate statistical methods of principal component analysis (PCA). Then linear discriminant analysis (LDA) was utilized to differentiate the loading score of different diseases as the diagnosing algorithm. Artificial neural network (ANN) was used for cross-validation. The diagnosis sensitivity and specificity by PCA-LDA are 88% and 79%, while that of the PCA-ANN are 89% and 95%. It can be seen that modern analyzing method is a useful tool for the analysis of serum spectra for diagnosing diseases.

In this paper, 514.5nm argon ion laser induced human serum Raman and auto-fluorescence spectra of normal, liver cirrhosis and liver cancer were measured and analyzed. The spectral differences between these three types of serums were observed and given brief explanations. Three parameters α, φ and Δλ were introduced to describe characteristics of each type of spectrum. Experimental results showed that these parameters might be applicable for discrimination of normal, liver cirrhosis and liver cancer, which will provide some reference values to explore the method of laser spectral diagnosis of cancer.

Surface enhanced Raman spectroscopy (SERS) has shown the advantage of detecting low concentration biofluids presently. Saliva SERS of 21 lung cancer patients and 22 normal people were measured and differentiated in this paper. Intensities of most peaks of lung cancer patients are weaker than that of normal people, some are stronger but with a small change rate. Those peaks were assigned to proteins and nucleic acids which indicate a corresponding decrease of substance in saliva. Principal component analysis (PCA) and linear discriminant analysis (LDA) were used to deduce and discriminate the two groups of data, resulted in accuracy, sensitivity, and specificity being 84%, 94%, and 81%, respectively. In conclusion, SERS of saliva has the ability of predicting lung cancer.

The spectral information, besides the spatial one, is very important in biology and medicine, as well as in many other areas. However the simultaneous analysis of the spatial and spectral information components is connected with certain difficulties which are caused primarily by the deficiencies of the devices which provide the spectral analysis (by light wavelengths) of images containing high spatial frequencies. We propose the method of biological objects images multispectral processing with rather high productivity. The device which provides this method performance includes a newly elaborated polychromic light source with real time controlled spectral composition for rough switching of the narrow spectral ranges, and acousto-optic tunable filter (AOTF) with wide angular aperture - for fine tuning of the selected sub-images wavelengths. The method and device practical configuration are considered and discussed. Also some features of AOTF required in the presented devices are analyzed. The possible information exchange between spectral and spatial information is also the subject of consideration as well the limitations of spectral and spatial resolving power. The experimental results connected with real time multispectral processing of tomographic images are presented and discussed. Also the possibilities of the method application for biology, medicine, and environment protection are considered.

Light scattering properties of bacterial cells mostly depend on their sizes, refractive indexes of their components and surrounding environment. Interaction between bacterial cells and 3d³ type transition metals causes their optical characteristics' changes. Desulfuromonas acetoxidans are uncolored gram-negative obligatory anaerobic sulfur reducing bacteria that can be used as microbial fuel cells with high electron recovery from different organic compounds oxidation to electric current as a result of electrons transfer in the processes of sulfur and some 3d³ type transition metals reduction, such as Ferrum and Manganese. In this study size distribution and relative content in the chosen interval of sizes (0.2 - 2.0 μm) of sulfur reducing D. acetoxidans bacterial cells under the influence of different concentrations of manganese chloride (II) hexahydrate, ferrous chloride (III) hexahydrate and ferrous sulfate (II) have been investigated by the new method of measuring. A method includes sounding of flow suspended bacterial cells by monochromatic coherent light, registration of signals of co-operation of sounding radiation with the explored microbiological objects by detects amplitudes and durations of scattered light impulses. Correlation between changes of light-scattering properties and growth of Desulfuromonas acetoxidans cells under these conditions has been shown.

Based on laser technology it is possible to visualize nanoparticles (NP) as light scattering objects by means of a conventional light microscope. Image series of visualized NPs were used to calculate characteristic parameters such as particle size and diffusion coefficient. Here progressive agglomeration of NP upon changes of ionic strength could be followed by repeated observation over minutes. While these analyses were based on dynamic properties of NPs, single particle paths were used to analyse particle agglomeration in more detail using image analysis methods. Together these techniques may help to better describe the behaviour of nanoparticles when used for cell culture experiments.

Raman spectroscopy of tissues has been widely studied for the diagnosis of various cancers, but biofluids were seldom used as the analyte because of the low concentration. Herein, serum of 30 normal people, 46 colon cancer, and 44 rectum cancer patients were measured Raman spectra and analyzed. The information of Raman peaks (intensity and width) and that of the fluorescence background (baseline function coefficients) were selected as parameters for statistical analysis. Principal component regression (PCR) and partial least square regression (PLSR) were used on the selected parameters separately to see the performance of the parameters. PCR performed better than PLSR in our spectral data. Then linear discriminant analysis (LDA) was used on the principal components (PCs) of the two regression method on the selected parameters, and a diagnostic accuracy of 88% and 83% were obtained. The conclusion is that the selected features can maintain the information of original spectra well and Raman spectroscopy of serum has the potential for the diagnosis of colorectal cancer.

The technology of laser-induced auto-fluorescence spectroscopy was used on serum for the diagnosis of lung cancer. We use principal component analysis and discriminant analysis to analyze spectra, and got an accuracy of 88% in distinguishing lung cancer patients and healthy people.

This paper presents the validation of a new multispectral camera specifically developed for dermatological application based on healthy participants from five different Skin PhotoTypes (SPT). The multispectral system provides images of the skin reflectance at different spectral bands, coupled with a neural network-based algorithm that reconstructs a hyperspectral cube of cutaneous data from a multispectral image. The flexibility of neural network based algorithm allows reconstruction at different wave ranges. The hyperspectral cube provides both high spectral and spatial information. The study population involves 150 healthy participants. The participants are classified based on their skin phototype according to the Fitzpatrick Scale and population covers five of the six types. The acquisition of a participant is performed at three body locations: two skin areas exposed to the sun (hand, face) and one area non exposed to the sun (lower back) and each is reconstructed at 3 different wave ranges. The validation is performed by comparing data acquired from a commercial spectrophotometer with the reconstructed spectrum obtained from averaging the hyperspectral cube. The comparison is calculated between 430 to 740 nm due to the limit of the spectrophotometer used. The results reveal that the multispectral camera is able to reconstruct hyperspectral cube with a goodness of fit coefficient superior to 0,997 for the average of all SPT for each location. The study reveals that the multispectral camera provides accurate reconstruction of hyperspectral cube which can be used for analysis of skin reflectance spectrum.

There exist numerous methods that aim to extract the optical parameters of a tissue by relating reflectance measurements to a theoretical model of light transport. During the parameter recovery process, assumptions are often made about the characteristics of the tissue. However, specious assumptions lead to inaccurate or even incorrect results. We present a method based on the maximum a posteriori estimation technique to recover the concentrations of the main chromophores present in a biological tissue from reflectance or transmittance measurements. The method provides correct results even in the presence of significant uncertainty in the underlying properties of the tissue. A preliminary analysis of the results obtained from simulated skin reflectance spectra suggests that the proposed MAP based method provides accurate estimates and is robust against a high level of uncertainty in the tissue's model. The results of phantom data are in agreement with the findings from our simulations as they emphasise the importance of including prior information about the unknown parameter in the estimation process.

An experimental RGB imaging system based on commercial color camera was constructed, and its potential for mapping of hemoglobin distribution in skin was studied. Two types of LEDs (RGB and white "warm" LEDs) were compared as illuminators for acquiring images of vascular and pigmented skin malformations. A novel approach for studies of skin capillary refill by RGB analysis has been proposed and discussed.

A novel fiber probe for spatially resolved reflectance measurements is presented, which uses simultaneously read-out spectrometers for each source-detector separation. Therefore, with this fiber probe and a Monte Carlo simulation, it is possible to determine spectrally resolved absorption and reduced scattering coefficients from various skinsites. The absolute calibration is done by using an integrating sphere but a phantom based calibration procedure was undertaken to compare the results of different calibration techniques. For tissue measurements, a standard SMA adaptor with a one inch diameter face can be used to provide a stable base for placing the probe onto the tissue and the possibility to apply pressure. The evaluation process was carried out by comparing the measured absorption and scattering of silicone and liquid phantoms to their reference values, obtained by integrating sphere spectroscopy. In addition, preliminary skin measurements are presented.

A basic problem intrinsic to many clinical diagnostic procedures as well as minimally invasive surgeries is the online invivo classification of tissue. Associated with this problem is the task to determine the boundaries between tissue sections of various degrees of disease progression, which cannot be identified easily. This problem is partly founded in the imaging modalities conventionally used, i.e., white-light endoscopy or fluorescence-based endoscopic imaging. These techniques allow for extracting of only a limited parameter set for judging the physiological or pathological state of tissue. Furthermore, fluorescence-based endoscopy relies on the administration of external labels, which principally disturbs the native tissue. These problems can be circumvented using Raman microspectroscopy as a diagnostic tool. Raman microscopy allows to record vibrational spectra at each sampling point. Therefore the molecular fingerprint of the sample can be deciphered with spatial resolution. It has been shown that Raman spectroscopy in combination with advanced statistical methods can be used to identify and grade tissue samples. However, the conventional approach of judging excised tissue sections by Raman microscopy does not present an approach which can be readily used in the clinics. Here we present our recent progress towards designing a fiber-based Raman probe, which - in perspective - might be incorporated into the working channel of a surgical endoscope. Thereby, it is anticipated to contribute to the clinical routine. We will review the general design principle of such a device and the specific design strategy for our Raman probe in concert with comparative measurements employing a set of home-built and commercially-available devices.

A photoplethysmography (PPG) signal can provide very useful information about a subject's hemodynamic status in a hospital or home environment. A newly developed portable multi-spectral photoplethysmography device has been used for studies of 11 healthy subjects. Multi-spectral photoplethysmography (MS-PPG) biosensor intended for analysis of peripheral blood volume pulsations at different vascular depths has been designed and experimentally tested. Multispectral monitoring was performed by means of a three-wavelengths (405 nm, 660 nm and 780 nm) laser diode and a single photodiode with multi-channel signal output processing. The proposed methodology and potential clinical applications are discussed.

The autofluorescence photobleaching intensity dynamics of in vivo skin and skin pathologies under continuous 532 nm laser irradiation have been studied. Overall the 141 human skin malformations were investigated by laser induced skin autofluorescence photobleaching analysis. Details of equipment are described along with some measurement results illustrating potentiality of the technology.

A clinical trial on multi-spectral imaging of malignant and non-malignant skin pathologies comprising 22 melanomas and 59 pigmented nevi was performed in Latvian Oncology Center. Analysis of data obtained in the spectral range 450-950 nm using multispectral camera have led to a novel image processing algorithm capable to distinguish melanoma from pigmented nevi and different areas of activity of melanoma. The proposed methodology and potential clinical applications are discussed.

Objective: The objective of this study was two folds: firstly, we would like to investigate the efficiency of bimodal spectroscopic technique in characterization of hypertrophic scarring tissue deliberately created on a preclinical model (rabbit's ear); on the other hand, we evaluate the inhibition effect of an anti-inflammatory medication (tacrolimus) on hypertrophic formation in scar by using our bimodal spectroscopic system. Study design: This study was conducted on 20 New Zealand Rabbits receiving hypertrophic scarring treatment on their ears. Fluorescence and Diffuse Reflectance spectra were collected from each scar, amongst which some had received tacrolimus treatment. Features were extracted from corrected spectral data and analyzed to classify the scarring tissues into hypertrophic or non-hypertrophic. Diagnostic algorithms were developed with the use of k-NN classifier and validated by comparing to histological classification result with Leave-one- out cross validation. Results and discussion: The accuracy of our bimodal spectroscopy method for detecting hypertrophic scarring scar tissue was good (sensibility: 90.84%, specificity: 94.44%). The features used for classification were mainly extracted from the spectra exited at 360, 410 and 420 nm. This indicates that the difference between the spectra acquired from hypertrophic and non-hypertrophic tissue may be due to the different intensity distribution of several fluorophores (collagen,elastin and NADH) excited in this range, or to the change in proportion of tissue layers (epidermis and dermis) explored by the CEFS in use.

Metal nanoparticles can function as optical contrast enhancers for reflectance-based diagnosis of epithelial precancer. We carry out Monte Carlo simulations to model photon propagation through normal tissues, unlabeled precancerous tissues, and precancerous tissues labeled with gold nanospheres and we compute the spectral reflectance response of these different tissue states. The results indicate that nanoparticle-induced changes in the spectral reflectance profile of tissues depend not only on the properties of these particles but also on the source-detector geometry used. When the source and detector fibers are oriented side by side and perpendicular to the tissue surface, the reflectance intensity of precancerous tissue is lower compared to that of normal tissue over the entire wavelength range simulated and addition of nanospheres enhances this negative contrast. When the fibers are tilted toward each other, the reflectance intensity of precancerous tissue is higher compared to that of normal tissue and labeling with nanospheres causes a significant enhancement of this positive contrast. The results also suggest that model-based spectral analysis of photon propagation through nanoparticle-labeled tissues provides a useful framework to quantify the extent of achievable contrast enhancement due to external labeling and to assess the diagnostic potential of nanoparticle-enhanced optical measurements.

Sentinel Lymph Node Biopsy (SLNB) is an increasingly standard procedure to help oncologists accurately stage cancers. It is performed as an alternative to full axillary lymph node dissection in breast cancer patients, reducing the risk of longterm health problems associated with lymph node removal. Intraoperative analysis is currently performed using touchprint cytology, which can introduce significant delay into the procedure. Spectral imaging is forming a multi-plane image where reflected intensities from a number of spectral bands are recorded at each pixel in the spatial plane. We investigate the possibility of using spectral imaging to assess sentinel lymph nodes of breast cancer patients with a view to eventually developing an optical technique that could significantly reduce the time required to perform this procedure. We investigate previously reported spectra of normal and metastatic tissue in the visible and near infrared region, using them as the basis of dummy spectral images. We analyse these images using the spectral angle map (SAM), a tool routinely used in other fields where spectral imaging is prevalent. We simulate random noise in these images in order to determine whether the SAM can discriminate between normal and metastatic pixels as the quality of the images deteriorates. We show that even in cases where noise levels are up to 20% of the maximum signal, the spectral angle map can distinguish healthy pixels from metastatic. We believe that this makes spectral imaging a good candidate for further study in the development of an optical SLNB.

A multi-spectral illumination approach for real-time mapping of the presence of gold nanoparticles in bulk tissue is presented. A Principal Component Analysis method is followed for determining the wavelengths that will make up the multispectral imaging endoscope.

Cancer is the second most cause of death in the world after cardiovascular related disease. This paper presents the calibration and test results obtained by mean of a hyperspectral reflectance and flexible video endoscope setup. Its application field is intended to be gastrointestinal cancer detection. We fabricated hard tissue phantoms which mimic different types of tissue in terms of its reflection properties for evaluation. The reflectance properties of the phantoms are set by varying the concentration of ink or titanium oxide. The goal is to achieve a similar reflectance properties as in actual respective tissues in vivo. A modified endoscope was used to discriminate the normal and tumor tissue phantoms with reflectance measurements. This hyperspectral endoscope setup consists of a light source, a camera and a camera controller that are compatible for use with conventional video endoscopes and video monitors. This setup allows the operator to switch between conventional white light imaging mode (WLI) and hyperspectral imaging mode (HSI). A significant imaging contrast between normal and tumor tissue phantoms has been provided.

We present a new method determining the absorption coefficients and the reduced scattering coefficients of in vivo rat cerebral cortex using single reflectance fiber probe with two source-collector geometries. Experiments with optical phantoms were conducted to evaluate the performance of the proposed fiber probe system. In order to confirm the possibility of the method to evaluate changes in the optical properties of cerebral cortex, we performed in vivo experiments for exposed rat brain during CSD evoked by the pinprick stimulation.

To characterize the degree of consistency of parameters of the optically uniaxial birefringent protein fibrils nets of biological tissues a new parameter - complex degree of mutual anisotropy is suggested. The technique of polarization measuring the coordinate distributions of the complex degree of mutual anisotropy of biological tissues is developed. It is shown that statistic approach to the analysis of complex degree of mutual anisotropy distributions of skin derma of various optical thicknesses appears to be more sensitive and efficient in differentiation of physiological state in comparison with investigations of complex degree of mutual polarization of the corresponding laser images.

Wear debris produced from articulating prosthetic joints is thought to be phagocytosed by macrophages which then release pro-inflammatory cytokines leading to the eventual aseptic loosening of the implant. Currently it is difficult to image wear particles within cells due to the lack of suitable ways of introducing tag molecules into the materials. We report how coherent anti-Stokes Raman scattering (CARS) spectroscopy can be used to image unlabeled material within cells relying on inherent chemical contrast. Using model particles we show how CARS signals change with respect to size and environment of the scattering particle. Incubating particles of polystyrene, polymethylmethacrylate and polyethylene with RAW264.7 macrophage cells, we demonstrate that it is possible to image cells phagocyotosing particles as well as to characterize the location of particles in three dimensions using the inherent optical sectioning ability of CARS. These results suggest that CARS provides an important tool for monitoring the accumulation of wear debris generated from prosthetic implants.

Medical expertise is frequently elicited to aid in determining the age and the cause of the trauma or injury. Child protection and law enforcement frequently rely on the physical assessment of the trauma which involves delineating intentional from unintentional types of trauma. Recent studies have shown that current methods to assess the age of traumatic injuries are highly inaccurate and do not give reasonable predictions. Hemoglobin is one of the strongest chromophores in human tissues. Transport of hemoglobin and its breakdown products in tissue determines the spectrophotometric characteristics of the skin and its variations in time. Therefore, measurements of diffuse reflective spectra of the skin allow noninvasive screening. This paper reviews potential transmission and diffusive reflection spectroscopy based techniques and predictive and quantitative modeling methods assisting in efficient retrieval of the age of extravascular contusions. This paper then presents a novel Monte Carlo technique for 3D photon tracking and blood transport model. In future studies, clinically obtained spectra will be used to validate the model as well as fine-tune coefficients for absorption. It is the goal of this study to develop a model that would allow a non-invasive, accurate determination of the age of a bruise.

We present Monte Carlo simulations of the backscattering of polarized light by colon tissue in terms of Mueller matrix. We validated the Monte Carlo code with measurements on aqueous suspensions of polystyrene spheres of different sizes. In a first instance we have modeled a tissue as a monodisperse scattering medium representing the nuclei in cytoplasm; then we included a second layer with monodisperse scatterers to represent the most superficial layers (mucosa and submucosa) while the deeper layers (muscularis and pericolic tissue) were "lumped" into a totally depolarizing lambertian. These simulations failed to reproduce the Rayleigh type scattering (larger depolarization for circular vs. linear incident polarization) systematically observed on all experimentally studied tissue samples. This issue has been solved by modelling tissues as a single layer of bimodal mixtures including large and small scatterers over a lambertian.