This PDF file contains the front matter associated with SPIE Proceedings Volume 7368, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

A laser scanner ophthalmoscope was developed for in vivo fluorescence lifetime measurements at the human retina. Measurements were performed in 30 degree fundus images. The fundus was excited by pulses of 75 ps (FWHM). The dynamic fluorescence was detected in two spectral channels K1(490-560nm), K2(560-700 nm) by time-correlated single photon counting. The decay of fluorescence was three-exponentially. Local and global alterations in lifetimes were found between healthy subjects and patients suffering from age-related macular degeneration, diabetic retinopathy, and vessel occlusion. The lifetimes T1, T2, and T3 in both channels are changed to longer values in AMD and diabetic retinopathy in comparison with healthy subjects. The lifetime T2 in K1 is most sensitive to metabolic alterations in branch arterial vessel occlusion.

During clinical application of the fluorescence lifetime laser scanner ophthalmoscope, a stepped slope of ocular autofluorescence was found. Fitting of fluorescence results in wrong lifetimes if the left border of the fitting interval is set at the time channel of first appearance of fluorescence. A better fit was reached at least for the fluorescence decay if the left border of the fitting interval is set near the maximum of detected fluorescence. Analysing the appearance of the stepped slope, its origination was found by the different appearance time of fluorescence from the crystalline lens and from the fundus fluorescence. The extension of the exponential model function by a parameter tci results in an optimal fit of both the slope and of the fluorescence decay. This new parameter describes differences in the appearance time of fluorescence originating from different layers. Taking into account the refractive index between layers, the geometrical distance between them can be determined. In this way, functional information (lifetimes) and geometrical information (distances) can be determined by the same measurement. To reach geometrical resolution comparable with OCT, pulses and time resolution are required in the order of 30 fs.

This study aimed to investigate the feasibility of non-invasive evaluating vessel blood oxygenation in the human eye using a dual-wavelength confocal scanning laser ophthalmoscope (cSLO). A cSLO prototype was adapted to use a new combination of two red and infrared wavelengths simultaneously performing a spectral analysis of the status of retinal blood vessel to give the ability to image perfusion in the human eye. This technique was evaluated using measurements made on normal volunteers' eyes. The reproducibility of the measurements of reflected light from the eye was investigated. Spectral measurements of light reflected from retinal vessels were determined and liner relationship of oxygen saturation of blood and spectral transmittance based on Beer-Lambert law was defined. The relative oxygen levels of retinal blood vessels (artery and vein) were successfully determined using these two wavelengths. The technique showed promise in the determination of the relative oxygen saturation levels of the retina in amongst eye subjects.

Imaging modalities like hyperspectral imaging create large amounts of data. Time efficient, automated analytic techniques are therefore required to enjoy the power of such methods. In this study it was investigated if hyperspectral imaging followed by automated noise filtering and statistical image analysis is a suitable method for characterization of the macroscopic structure of atherosclerotic lesions. Ten human aorta samples (6×8 cm) were collected during autopsy. Hyperspectral white light and fluorescence images and 5 - 6 biopsies were collected from each sample. The biopsies were stained (HES, Sudan red), and grouped according to histology. All images were noise filtered and normalized. Fluorescence spectra were collected from all biopsied regions, and used to compute average spectra for each histological group. Supervised classification was performed using Spectral angle mapping (SAM) with the average spectra as endmembers. K-means- and ISO-data clustering was used for unsupervised classification. The results show that noise filtering and normalization is essential for reliable classification. Supervised classification was in general found to perform better than unsupervised classification. However, the SAM results strongly depend on the variation in the spectra used to compute the average endmember spectra. The analysis show that fatty deposits, calcifications, connective tissue and hemoglobin can be identified. The lesions were found to have a complex structure where vulnerable regions could be found next to stabile regions within the same lesion. In conclusion hyperspectral imaging, automated filtering and -analysis was found to be a suitable tool to classify advanced atherosclerotic lesions.

Raman signals of biological tissues are distorted by the influence of tissue absorption and scattering properties, which significantly challenges signal quantification. We investigated the influence of the tissue optical properties on the resonance Raman signal of β-carotene with tissue phantom measurements and Monte Carlo simulations. Both methods show that the Raman signal drops roughly proportional to 1/μ_a for absorption coefficients (μa) in the range of 0.1-4mm^-1. The influence of the reduced scattering coefficient in the range of 0.1-8mm^-1 is less strong, but not negligible. We introduce combined Raman and spatially resolved reflectance measurements to measure both Raman signals and tissue optical properties. The optical properties were deduced from the spatially resolved reflectance measurements by a Monte Carlo-based lookup table (LUT). For the signal correction we applied correction functions derived from the Monte Carlo simulations. We corrected in vivo resonance Raman measurements of carotenoids in caucasian skin (n=9) with respect to the mean optical properties of the group. The magnitude of the average correction effect was found to be 18±11% on the minimal pigmented palm.

The Multispectral Dermoscope has been used for imaging skin lesions. Illumination at three different spectral regions and subsequent image processing can provide information on the localization of melanin, hemoglobin and scattering structures in the skin. The multispectral dermoscope was used for the diagnoses of skin lesions and to monitor the effect of Pulsed Dye Laser treatment on skin lesions.

Spatially-resolved bimodal spectroscopy (multiple AutoFluorescence AF excitation and Diffuse Reflectance DR), was used in vivo to discriminate various healthy and precancerous skin stages in a pre-clinical model (UV-irradiated mouse): Compensatory Hyperplasia CH, Atypical Hyperplasia AH and Dysplasia D. A specific data preprocessing scheme was applied to intensity spectra (filtering, spectral correction and intensity normalization), and several sets of spectral characteristics were automatically extracted and selected based on their discrimination power, statistically tested for every pair-wise comparison of histological classes. Data reduction with Principal Components Analysis (PCA) was performed and 3 classification methods were implemented (k-NN, LDA and SVM), in order to compare diagnostic performance of each method. Diagnostic performance was studied and assessed in terms of Sensibility (Se) and Specificity (Sp) as a function of the selected features, of the combinations of 3 different inter-fibres distances and of the numbers of principal components, such that: Se and Sp ≈ 100% when discriminating CH vs. others; Sp ≈ 100% and Se > 95% when discriminating Healthy vs. AH or D; Sp ≈ 74% and Se ≈ 63% for AH vs. D.

An experimental set-up is presented for wavelength and spatially resolved reflectance measurements (SRR) via optical fibers and imaging optics. In order to characterize the set-up, we made a set of silicone rubber phantoms with different absorber and scatterer concentrations. The remission profiles gained from the phantoms are the input for a look-up table (LUT) based "inverse Monte-Carlo simulation" to deduce absorption μ_a and reduced scattering coefficients μ_s'. As an independent method for determination of μ_a and μ_s' we also made measurements with an 'integrating sphere spectrometer' (ISS). Our normalization procedure of the SRR measurements is presented and the validity of this method is discussed.

In this contribution we present the application of five different commercially available semiconductor core/shell quantum dots (Qdot® Nanocrystals - Invitrogen Corp.) as multiplexing FRET acceptors together with a commercial supramolecular terbium complex (Lumi4®-Tb - Lumiphore Inc.) as donor in a homogeneous immunoassay format. To realize the molecular recognition necessary for a FRET assay, the terbium complex was labeled to streptavidin (sAv- Lumi4-Tb) and the QDs were surface functionalized with biotin (Biot-QD). The biotin-streptavidin binding serves as a proof-of-principle representative for many biological interactions taking place on the nanometer scale (e.g. immunoassays). The presented FRET system can be efficiently used for the detection of inter- and intramolecular processes for clinical diagnostics and biomedical spectroscopy as well as molecular ruler applications and microscopy.

A membrane associated caspase sensor based on Förster Resonance Energy Transfer (FRET) between enhanced cyan fluorescent protein (Mem-ECFP) and yellow fluorescent protein (EYFP) is reported. Upon apoptosis a caspase sensitive amino acid peptide linker (DEVD) between these proteins is cleaved, and pronounced changes of fluorescence spectra and lifetimes are observed. Membrane selective detection of fluorescent proteins in cultivated HeLa cervix carcinoma cells is achieved by total internal reflection fluorescence microscopy (TIRFM) with high sensitivity and resolution.

The application of surface enhanced Raman spectroscopy in combination with a microfluidic device and an isotopeedited internal standard seems to be a promising way for a new approach for quantitative SERS measurements. An innovative lab on a chip system offers the possibility for reproducible, quantitative online SERS measurements based on the application of isotope labelled internal standards and liquid/liquid segmented flow based flow-through Raman detection. Errors caused by the used method can be compensated by using an internal standard.

Urinary tract infection diagnosis and antibiogram require a 48 hour waiting period using conventional methods. This results in ineffective treatments, increased costs and most importantly in increased resistance to antibiotics. In this work, a novel method for classifying bacteria and determining their sensitivity to an antibiotic using Raman spectroscopy is described. Raman spectra of three species of gram negative Enterobacteria, most commonly responsible for urinary tract infections, were collected. The study included 25 samples each of E.coli, Klebsiella p. and Proteus spp. A novel algorithm based on spectral ratios followed by discriminant analysis resulted in classification with over 94% accuracy. Sensitivity and specificity for the three types of bacteria ranged from 88-100%. For the development of an antibiogram, bacterial samples were treated with the antibiotic ciprofloxacin to which they were all sensitive. Sensitivity to the antibiotic was evident after analysis of the Raman signatures of bacteria treated or not treated with this antibiotic as early as two hours after exposure. This technique can lead to the development of new technology for urinary tract infection diagnosis and antibiogram with same day results, bypassing urine cultures and avoiding all undesirable consequences of current practice.

In cell biology one of the most important but also most difficult tasks is to visualize dynamic 3-dimensional intracellular processes and alterations. In combination with an ordinary microscope, digital holography provides contact-less, marker-free, quantitative phase contrast imaging to record both long term time-lapse investigations in toxicology and cancer research and fast dynamic processes like shape variations. However, the rather homogenous cellular refraction index limits the technology in case of the imaging of intracellular structures and processes. Here we demonstrate the first results of the selective alteration of the cytoplasmatic refraction index in order to enhance the intracellular contrast. Therefore we combined a commercial inverted microscope with the digital holography setup and a microinjection unit. The microinjection unit was used to inject a small amount of glycerol (50 % in water) to increase the intracellular refraction index. The injection process, the flow, the spreading and the final mixing of glycerol with the surrounding medium could be visualized by digital holography. Non diffractive reconstruction algorithms enable digital holographic focus adjustment with constant imaging scale, multi focus imaging of the object planes and subsequent focus correction. We injected adherent A549 tumor cells in a perinuclear region with the glycerol-water mixture. The reconstructed images indicate an impermeability of the nuclear membrane for glycerol resulting in an enhanced intracellular contrast. The specific alteration of the intracellular refraction index allows the development of a 3-dimensional imaging for dynamic intracellular processes in living cells.

A dual-wavelength reflectance optical sensor for monitoring cutaneous blood perfusion is presented as a part of multisensor glucose monitoring system. A Monte-Carlo simulation of partial differential pathlengths has been used for the optimization of the distance from light source to detector. The simulation indicated that the light pathlength within the upper vascularised skin layers increases before reaching saturation at separation distances larger than 3 mm. Thus the sensor sensitivity does not benefit from larger source-detector distances. At the same time with a higher separation of the detector from the source, the intensity exponentially decreases while undesirable sensitivity to the muscle perfusion increases. The hardware prototype has been developed based on the simulation findings and tested in a laboratory setting and in a home use study by patients with diabetes. For both testing procedures the optical sensor demonstrated high sensitivity to perfusion changes. The effect of initial cutaneous blood increase under the sensor has been observed which can be associated with pressure-induced vasodilation as a response to the sensor application.

An evaluation of the optimum choice of wavelengths, when using the 'Modified Lambert-Beer law' to estimate blood oxygen saturation, that minimises the mean error across a range of oxygen saturation values is presented. The stability of this approach and its susceptibility to noise are also considered.

MLL cells were incubated with Pd-porphyrin and irradiated at 405 nm. The change in Pd-porphyrin phosphorescence intensity was monitored during treatment. A time dependent diffusion model has been developed to fit the measured phosphorescence signals so that the value of the oxygen diffusion coefficient in cells can be calculated

We demonstrate that a colour RGB-CCD camera can be used to map haemoglobin changes of the exposed cortex following cortical activation and spreading depression in rats. We examine its performance by a comparison with narrow bandpass spectroscopy and evaluation of the extinction spectra of haemoglobin based on condition number analysis.

We used spatially resolved near-infrared spectroscopy (SRS-NIRS) to assess calf and thigh muscle oxygenation during running on a motor-driven treadmill. Two protocols were used: An incremental speed protocol (velocity = 6 - 12 km/h, ▵v = 2 km/h) was performed in 3 minute stages, while a pacing paradigm modulated step frequency alternatively (2.3 Hz [S_Low]; 3.3 Hz [SHigh]) during a constant velocity for 2 minutes each. A SRS-NIRS broadband system (600 - 1000 nm) was used to measure total haemoglobin concentration and oxygen saturation (SO2). An accelerometer was placed on the hip joints to measure limb acceleration through the experiment. The data showed that the calf (SO2 58 to 42%) desaturated to a significantly lower level than the thigh (61 to 54%). During the pacing protocol, SO2 was significantly different between the S_Low vs. S_High trials. Additionally, physiological data as measured by spirometry were different between the S_Low vs. SHigh pacing trials (VO2 (2563± 586 vs. 2503 ± 605 mL/min). Significant differences in VO2 at the same workload (speed) indicate alterations in mechanical efficiency. These data suggest that SRS broadband NIRS can be used to discern small changes in muscle oxygenation, making this device useful for metabolic exercise studies in addition to spirometry and movement monitoring by accelerometers.

We developed a compact dual-wavelength dual-channel system for time-resolved diffuse NIR spectroscopy that uses a novel approach based on space-multiplexing (instead of time-multiplexing) of wavelengths, to increase the signal-tonoise ratio and avoid cross-talk.

We assess the data quality of calculated tissue oxygen saturation (SO2) and haemoglobin concentrations recorded on muscle during an incremental cycling protocol in healthy volunteers. The protocol was repeated three times at the same day and a fourth time at a different day to estimate the reproducibility of the method. A novel broad-band, spatially resolved spectrometer (SRS) system was employed which allowed us to compare SRS-based oxygenation parameters with modified Lambert-Beer (MLB) data. We found that the inter-subject variation in SO₂ (standard deviation about 6 %) is considerably larger than the reproducibility (about 1.5 %) both for same day and different day tests. When changes in SO₂ during the cycling test were considered the reproducibility is better than 1 %. Time courses of SRS-based haemoglobin parameters are different from MLB-data with higher reproducibility for SRS. The magnitudes of the haemoglobin changes were found to be considerably larger for the SRS method. Furthermore, the broad band approach was tested against a four-wavelength analysis with the differences found to be negligible.

Tissue morphology, light attenuation and texture are analyzed from images acquired by swept-source Fourier-domain optical coherence tomography from arterial samples. The data were corrected for the effect of the confocal point spread function and were analyzed using the single scattering model.

A set-up for time-resolved transmittance and reflectance spectroscopy of diffusive media was upgraded to allow measurements to be performed continuously from 600 to 1100 nm. Time-resolved diffuse optical spectroscopy of breast was carried out on 10 healthy volunteers, demonstrating the feasibility of in vivo measurements up to 1100 nm. The optical characterization of collagen was also extended revealing an absorption peak (around 1020-1030 nm), which could prove of interest for the in vivo quantification of collagen.

An automated algorithm and methodology is presented to pathologically classify the scattering changes encountered in the raster scanning of normal and tumor pancreatic tissues using microsampling reflectance spectroscopy. A quasiconfocal reflectance imaging system was used to directly measure the tissue scatter reflectance in situ, and the spectrum was used to identify the scattering power, amplitude and total wavelength-integrated intensity. Pancreatic tumor and normal samples were characterized using the instrument and subtle changes in the scatter signal were encountered within regions of each sample. Discrimination between normal vs. tumor tissue was readily performed using an Artificial Neural Network (ANN) classifier algorithm. A similar approach has worked also for regions of tumor morphology when statistical pre-processing of the scattering parameters was included to create additional data features. This automated interpretation methodology can provide a tool for guiding surgical resection in areas where microscopy imaging do not reach enough contrast to assist the surgeon.

The interconnection between geometry of biotissue structure with their polarization properties has been studied. It has been shown that for physiologically normal biotissues polarization properties of radiation scattered on architectonic nets formed by protein fibrils possess the fractal character. Pathological changes of biotissues architectonics are accompanied with the transformation of self-similar structure of Mueller-matrix images into stochastic and statistic ones.

A prototype clinical fluorescence and reflectance spectrometer was developed and employed to detect human pancreatic adenocarcinoma. For the first time, quantitative pancreatic tissue models and chemometric algorithms were applied to successfully distinguish among tissue types.

Fluorescence cystoscopy has been recently acknowledged as a useful method to detect early superficial bladder cancer, even flat lesions. After the instillation of hexaminolevulinic acid (Hexvix®) in the bladder for about an hour, photoactivable porphyrins (PaP), mainly protoporphyrin IX (PpIX) accumulate in the cancerous cells. Although we observe a selective production of PpIX and an outstanding sensitivity of this method, false positive (FP) lesions negatively impact its specificity. Carcinogenesis often combines with angiogenesis, and thus changes in vascular architecture. Therefore, the visualization of the vascular modifications on the fluorescence positive sites is likely to differentiate false and true positive (TP). New methods including high magnification (HM) cystoscopy are being investigated by our group, and will yield a reduced number of biopsies and a better characterization of the fluorescence positive sites. In this study, we are using a dedicated rigid cystoscope, allowing conventional magnification during "macroscopic" observation, as well as image acquisition with HM when the endoscope is in contact with the tissue. Each observed site is biopsied and described by histopathological analysis. The vascular organization (tortuosity, vascular loops, vascular area and diameter) of the fluorescence positive sites was characterized in situ. Intrinsic contrast between the vessels and the tissue was enhanced with an optimization of the spectral design. Preliminary results are presented here.

Prostate cancer is a common disease among men with an increasing number of incidences during the last three decades. Histopathological grading of prostate cancer is based on tissue structural abnormalities. Gleason grading system is the gold standard and is based on the organization features of prostatic glands. However, till now there is an uncertainty assign Gleason grade to intermediate stages of the disease, Gleason 3 and Gleason 4. The aim of this study was to explore the possibility of introducing fluorescent probes in this prostate cancer Gleason grading problem. Propidium Iodide with cellular nuclei binding pattern and Alexa 488-WGA with selectivity in polysaccharides with sialic acid residues were finally chosen. Their localisation patterns were assessed using confocal microscopy. Their colocalisation degree was quantified using special developed algorithms of image processing and analysis. The introduced metrics of colocalisation were successfully used to correct classify samples in Gleason 3 and Gleason 4 grades. These metrics were found appropriate to correctly classify 93.10 % of the images into the two classes using the logistic algorithm. The integration of confocal microscopy along with fluorescent probes to pathologist routine, is an approach that cloud lead to prognostic advances.

A method was developed to estimate spectral changes of the absorption properties of turbid media from time-resolved reflectance/transmittance measurements. It was derived directly from the microscopic Beer-Lambert law, and tested against simulations and phantom measurements.

The interrelation of orientation, anisotropy structure of biological tissue architectonics and topological element distribution of John's Matrices is investigated here. It is researched the analytical correlation of bioobject John's matrices microstructure with matrix element indices measured in the far field of Fraunhofer's diffraction. The investigation is also dealt with the computer modeling and experimental researching the structure of matrix operator of multifractal amorphous - crystalline organization of different morphological structure biological tissues.

There have been theoretically analyzed the ways of the formation of the polarization singularities of the biological tissues images of various morphological structures. There have been also experimentally examined the coordinate distributions of a single and doubly degenerated polarization singularities of the physiologically normal and pathologically changed biological tissues. There have been determined the statistical criteria of diagnostics of the kidney tissue collagenous disease (the 3rd and the 4th statistical moments of the linear density singularity points). It was found out that the process of the pathological change of the kidney tissue morphology leads to the formation of the self-similar (fractal) distribution of the polarization singularities of its image.

We studied the angular distribution of remitted and reflected light from rough turbid biological media. The remission and reflection are studied separately, then they are compared with each other. The angular distribution of the reflection/remission from 'reflecting standards' is investigated.

Visible integrating sphere spectroscopy was applied to determine the optical properties of turbid media in the spectral range of 480 to 650 nm. Total transmission and diffuse remission were measured. As samples, we used liquid phantoms consisting of pH-buffer solution and different amounts of Lipovenoes 10% or black ink. It was investigated if the scattering coefficient and the absorption coefficient of turbid media can be accurately obtained.

In vivo studies of single molecule dynamics by means of Fluorescence correlation spectroscopy can suffer from high background. Fluorescence lifetime correlation spectroscopy provides a tool to distinguish between signal and unwanted contributions via lifetime separation. By studying the motion of the RNA-induced silencing complex (RISC) within two compartments of a human cell, the nucleus and the cytoplasm, we observed clear differences in concentration as well as mobility of the protein complex between those two locations. Especially in the nucleus, where the fluorescence signal is very weak, a correction for background is crucial to provide reliable results of the particle number. Utilizing the fluorescent lifetime of the different contributions, we show that it is possible to distinguish between the fluorescent signal and the autofluorescent background in vivo in a single measurement.

Diameters of single polystyrene beads were determined within ~10 nm accuracy by comparing Mie theory oscillations and wavelength resolved measurements. The setup is realized with an axicon supported reflected darkfield microscope and is herein presented in detail. Further we explain a theoretical model considering the effective numerical aperature of the measuring system. A fitting algorithm allows rapid characterization of the sphere diameters.

A flexible optical fiber probe for attenuated total reflection (ATR) spectroscopy with Fourier-transfer infrared (FT-IR) spectroscope is developed. The probe is based on a hollow optical-fiber that comprises a polycarbonate thin tube and silver- and polymer-inner layers and that shows low losses in a wide wavelength range in the infrared. The probe has an ATR prism whose shape is optimized for use with hollow optical fibers. Some results on preliminary experiments show the potential uses of the probe in clinical applications. The probe is appropriate for in-vivo applications used with a thin endoscope because the fiber comprises only non-toxic and chemically durable materials.

This paper describes a new multifunctional laser noninvasive diagnostic system (MLNDS) for medicine. In a single hardware MLNDS combines 3 different in vivo laser diagnostic techniques: Laser Doppler Flowmetry, Laser Fluorescent Diagnostics and Reflectance Tissue Oximetry. All these methods together allow a doctor to evaluate more exactly and in vivo a functional condition of soft tissues, especially to study the finenesses of respiratory and blood microcirculation processes in a skin and mucosa. The complex complementary diagnostics turns out to be more powerful than a trivial sum of isolated one. To produce more precise measurements a number of problems of metrological providing for that have been studied as well as a set of simple, reproducible and photostable calibration gauges with tissue-like optical properties has been created.

IR absorption spectra of urea, urine without any deviation in composition and kidney oxalate calculi are studied. The shifting of stretching vibrations of carbonyl group C=O confirms that assert that the joining of calcium oxalate molecules and oxalate complexes join to urea molecules via oxygen atom of urea. The shifting of COOsymmetric stretching vibration frequencies may testify the presence of molecules and aggregates of calcium oxalates. Obtained results may be useful in developing new methods in early diagnostics of different diseases.

A transillumination breast spectroscopy (TiBS) system used for breast cancer risk assessment is being modified to facilitate large-scale trials and to simply use. A proposed change involves switching from a broadband light source to several laser sources cycled through in sequence, which will allow for a wavelength-independent detection system. The effect of the reduction of the spectral content of the system on the ability to predict mammographic density (a known breast cancer risk factor) from the spectra was assessed. Wavelengths for the laser sources were chosen based on their contribution to the loading vectors obtained from a principal components analysis of spectra from a study correlating TiBS spectra with mammographic density. 12 wavelengths were selected based on the principal component loads. Principal component scores were obtained using both full-spectrum and 12-wavelength-spectrum data. No significant loss of predictive ability was found when the broadband spectra were reduced to only 12 wavelengths-for both data sets, 3 principal component scores were significantly able to distinguish between high- and low-mammographic density groups.

Laser surgery gives the possibility to work remotely which leads to high precision, little trauma and high level sterility. However these advantages are coming with the lack of haptic feedback during the laser ablation of tissue. Therefore additional means are required to control tissue-specific ablation during laser surgery supporting the surgeon regardless of experience and skills. Diffuse Reflectance Spectroscopy provides a straightforward and simple approach for optical tissue differentiation. We measured diffuse reflectance from four various tissue types ex vivo. We applied Linear Discriminant Analysis (LDA) to differentiate the four tissue types and computed the area under the ROC curve (AUC). Special emphasis was taken on the identification of nerve as the most crucial tissue for maxillofacial surgery. The results show a promise for differentiating soft tissues as guidance for tissue-specific laser surgery by means of the diffuse reflectance.

Titanium dioxide (TiO₂) nanoparticles are extensively used nowadays in sunscreens as protective compounds for human skin from UV radiation. In this paper, such particles are investigated from the viewpoint of penetration into living skin, UV protective properties (compared with silicon (Si) particles) and as sources of free radicals if UV-irradiated. We show that: a) even after multiple applications, the particles are located within the uppermost 3-μm-thick part of the skin; b) the optimal sizes are found to be 62 nm and 55 nm, respectively for TiO₂ and Si particles for 310-nm light and, correspondingly, 122 and 70 nm - for 400-nm radiation; c) if applied onto glass, small particles of 25 nm in diameter produce an increased amount of free radicals compared to the larger ones of 400 nm in diameter and placebo itself; however, if applied onto porcine skin in vitro, there is no statistically distinct difference in the amount of radicals generated by the two kinds of particles on skin and by the skin itself. This proves that although particles as part of sunscreens produce free radicals, the effect is negligible in comparison to the production of radicals by skin in vitro.

Fluorescent analysis of basal cell carcinoma (BCC), squamous cell carcinoma (SCC), keratoacanthoma and benign cutaneous lesions is carried out under initial phase of clinical trial in the National Oncological Center - Sofia. Excitation sources with maximum of emission at 365, 380, 405, 450 and 630 nm are applied for better differentiation between nonmelanoma malignant cutaneous lesions fluorescence and spectral discrimination from the benign pathologies. Major spectral features are addressed and diagnostic discrimination algorithms based on lesions' emission properties are proposed. The diagnostic algorithms and evaluation procedures found will be applied for development of an optical biopsy clinical system for skin cancer detection in the frames of National Oncological Center and other university hospital dermatological departments in our country.

In the recent study delta-ALA/PpIX is used as fluorescent marker for dysplasia and tumor detection in esophagus, stomach and colon. ALA is administered per os six to eight (depending on the lesion location) hours before measurements at dose 20mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built for the LED to use the light guide of standard video-endoscopic system. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. The fluorescence detected from tumor sites has very complex spectral origins. It consists of autofluorescence, fluorescence from exogenous fluorophores and re-absorption from the chromophores accumulated in the tissue investigated. Spectral features observed during endoscopic investigations could be distinct as the next regions: 450-630 nm region, where tissue autofluorescence is observed; 630-710 nm region, where fluorescence of PpIX is clearly pronounced; 530-580 nm region, where minima in the autofluorescence signal are observed, related to re-absorption of oxy-hemoglobin in this spectral area. Endogenous and exogenous fluorescence spectra are used to develop simple but effective algorithm, based on dimensionless ratio of the signals at 560 and 635 nm, for differentiation of normal/abnormal gastrointestinal tissues. Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

Using planar waveguides as a platform for optical biosensors allows an efficient and selective fluorescence excitation in close proximity to the waveguide surface. Usually, the fluorescence light that is emitted in the space above the sensor chip is collected and analyzed by suitable free space optics and a detector. Due to the vicinity of the fluorescent molecules to the interface of the waveguide layer, a substantial part of the fluorescence light is coupled back into and collected by the waveguide. The coupling efficiency depends on position, environment and orientation of the molecules. The utilization of this signal for fluorescence detection and analysis can allow a significant simplification of the optical instrumentation. We present a fundamental investigation of the fluorescence collection efficiency into the waveguide by theoretical and experimental means.

Sterility testing of cell or tissue cultures is an essential task in the fields of regenerative medicine and tissue engineering. Especially in case of Good manufacturing practice (GMP) of cell and tissue based transplants. We present a system based on a commercially available microscope equipped with a microfluidic cell that prepares the particles found in the solution for analysis. A Raman-spectrometer attachment optimized for non-destructive, rapid recording of Raman spectra, and a data acquisition and analysis tool for identification of the particles. Identification of critical particles like microorganisms via microscopic imaging and subsequent image analysis is carried out before micro-Raman analysis of those particles is then carried out with an excitation wavelength of 785 nm. However an automated image analysis of small particles from supernatant of biopsies on a filter chip with tiny holes is a difficult task. Especially for the discrimination of small particles like cell debris and bacteria, which have a quite similar range of size. Because of that particles in the supernatant and microorganisms have to be discriminated by means of Raman spectroscopy. We present here a Raman based method to discriminate between cells, microorganisms and particles in cell culture.

The estimation of the nutritional parameters of food products is a difficult and laborious process. Many companies spend considerable financial and other resources to frequently check the nutritional facts of their products. In addition, current methods are unsuitable for day-to-day, restaurant or home use. A new device, that would automatically estimate the nutritional facts of any edible product, could prove very useful in all of the above situations. To achieve that goal, Raman Spectroscopy was used to examine a wide variety of commonly available food products. There was minimal sample preparation, mainly homogenization and dilution. Raman spectra were collected with 785 nm excitation and 4.5 cm^-1 resolution. The spectra were analyzed and solutions to linear differential equations resulted in estimates of nutritional facts. When the analysis techniques were optimized, several nutritional parameters could be estimated, such as calories, fat, protein, carbohydrates, sugars, and fiber, with an error between 2.9 % and 6.7 %. The results imply that Raman spectroscopy can be used for the estimation of the nutritional facts of food products with an error less than what is required for labeling. A device based on this technique could prove to be a very useful tool for dieticians, hospitals, food companies, health care organizations, restaurants and even home users, who want to be informed about the content of the food that they consume.

Changes in scalp and cortical blood flow induced by voluntary hyperventilation are investigated by near-infrared diffusing-wave spectroscopy. The temporal intensity autocorrelation function g(²) (τ) of multiply scattered light is recorded from the forehead of subjects during hyperventilation. Blood flow within the sampled tissue volume is estimated by the mean decay rate of g(²) (τ) . Data measured from six subjects show that the pattern of the hemodynamic response during 50 s hyperventilation is rather complicated: within the first 10 s, in three subjects an initial increase in blood flow is observed; from 10 s to 20 s, the mean blood flow is smaller than its baseline value for all six subjects; for the duration from 20 s to 30 s, the blood flow increases again. However, after 30 s the change is not consistent across subjects. Further study on one of these subjects by using two receivers probing the blood flow in the cortex and in the superficial layers simultaneously, reveals that during hyperventilation, the direction of change in blood flow within the scalp is opposite to the one in the brain. This helps to understand the complicated hemodynamic response observed in our measurements.

Medical near-infrared spectroscopy (NIRS) can be used to estimate cerebral haemodynamic changes non-invasively. Sleep apnea is a common sleep disorder where repetitive pauses in breathing decrease the quality of sleep and exposes the individual to various health problems. We have measured oxygenated and deoxygenated haemoglobin concentration changes during apneic events in sleep from the forehead of one subject using NIRS and used principal component analysis to extract extracerebral and cortical haemodynamic changes from NIRS signals. Comparison of NIRS signals with EEG, bioimpedance, and pulse oximetry data suggests that termination of apnea leads to decreases in cerebral blood volume and flow that may be related to neurological arousal via neurovascular coupling.

The arterial inflow and the venous capacitance in the human skin were visualized from the increase rate and the change of total blood concentration derived from RGB images during upper limb occlusion at 50 mmHg-pressure.