Employing a reflectance multi-photon microscopy (MPM) technique, we developed novel method to quantitatively study the three-dimensional assembly of structural proteins within bulk of dermal ECMs. Using a structurally simplified model of skin with enzymatically dissected epidermis, we find that low resolution MPM clearly discriminates between normal and pathological dermis. High-resolution images revealed that the backscattered MPM signals are affected by the assembly of collagen fibrils and fibers within this system. Exposure of tissues to high concentrations of potentially denaturing chemicals also resulted in the reduction of SHG signals from structural proteins which coincided with the appearance of aggregated fluorescent structures.

We used combined two photon intrinsic fluorescence (TPE), second harmonic generation microscopy (SHG), fluorescence lifetime imaging microscopy (FLIM), and multispectral two photon emission detection (MTPE) to investigate different kinds of human cutaneous ex-vivo skin lesions. Morphological and spectroscopic analyses allowed to characterize both healthy and pathological skin samples, including tumors, as well as to discriminate between healthy and diseased tissue, in a good agreement with common routine histology. In particular, we examined tissue samples from normal and pathological scar tissue (keloid), and skin tumors, including basal cell carcinoma (BCC) and malignant melanoma (MM). By using combined TPE-SHG microscopy we investigated morphological features of different skin regions, as BCC, tumor-stroma interface, healthy dermis, fibroblastic proliferation, and keloids. The SHG to autofluorescence aging index of dermis (SAAID) score was used to characterize each region, finding differences between BCC, healthy skin, tumor-stroma interface, keloids, and fibroblastic proliferation. Further comparative analysis of healthy skin and neoplastic samples was performed using FLIM. In particular, BCC showed a blue-shifted fluorescence emission, a higher absorption at 800 nm excitation wavelength, and a slightly longer mean fluorescence lifetime. MM showed a lifetime distribution similar to the corresponding melanocytic nevus (MN) lifetime distribution for the slow lifetime component, and different for the fast lifetime component.

Prostate cancer is a common malignancy among maturing men and the second leading cause of cancer death in USA. 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. Although Gleason score has contributed on cancer prognosis and on treatment planning, its accuracy is about 58%, with this percentage to be lower in GG2, GG3 and GG5 grading. On the other hand it is strongly affected by "inter- and intra observer variations", making the whole process very subjective. Therefore, there is need for the development of grading tools based on imaging and computer vision techniques for a more accurate prostate cancer prognosis. The aim of this paper is the development of a novel method for objective grading of biopsy specimen in order to support histopathological prognosis of the tumor. This new method is based on texture analysis techniques, and particularly on Gray Level Co-occurrence Matrix (GLCM) that estimates image properties related to second order statistics. Histopathological images of prostate cancer, from Gleason grade2 to Gleason grade 5, were acquired and subjected to image texture analysis. Thirteen texture characteristics were calculated from this matrix as they were proposed by Haralick. Using stepwise variable selection, a subset of four characteristics were selected and used for the description and classification of each image field. The selected characteristics profile was used for grading the specimen with the multiparameter statistical method of multiple logistic discrimination analysis. The subset of these characteristics provided 87% correct grading of the specimens. The addition of any of the remaining characteristics did not improve significantly the diagnostic ability of the method. This study demonstrated that texture analysis techniques could provide valuable grading decision support to the pathologists, concerning prostate cancer prognosis.

Raman imaging methods provides visual information of pathological changes of tissues and cells without labeling procedures. We are developing direct Raman imaging technique in order to measure a Raman image of living tissues and cells in a short period without mapping procedure. A preliminary system is prepared consisting of a background-free tunable laser, band-pass filters and a CCD detector. In the present study, it is demonstrated the viability of the technique in a real time measurement of the direct Raman images.

Solitary rectal ulcer syndrome (SRUS) is a chronic disease of the rectum. Although SRUS is a benign condition there are studies which suggest that chronic ischaemia which occurs in the SRUS may lead to "transitional mucosa" that is similar to that adjacent to colorectal carcinomas and adenomas and may lead to colorectal dysplasia and carcinoma development. The exclusion of primary or metastatic malignancy is the most important aim in the differential diagnosis of SRUS. In our study we assess the possibilities of autofluorescence colonoscopy (AFC) in diagnosis and management of SRUS. We performed white light colonoscopy first. The tissue samples were taken for pathological examination. When SRUS was histopathologically confirmed AFC was performed by means of Xillix OncoLIFE. During AFC numerical colour value (NCV) of autofluorescence of SRUS lesions was noted. During 1946 colonoscopies eight persons were diagnosed as having solitary rectal ulcer syndrome. We did not observe autofluorescence increase in case of polipoid and flat ulcer lesions (NCV 0,39-0,67; mean 0,525) and little increase of autofluorescence in case of erythema lesion (NCV- 0,94). SRUS is a rare disorder of the rectum but it causes differential diagnosis problems. The most common reason for incorrect diagnosis are inadequate tissue specimens. AFC allows to reveal subtle areas within the lesions of more intense autofluorescence and localizes the potential cancer-transformating dysplasia. In this way the most representative area with highest risk of pre- or cancerous changes, for biopsy specimen is indicated.

A method is proposed for determining blood oxygen saturation in frozen tissue. The method is based on a spectral camera system equipped with an Acoustic-Optical-Tuneable-Filter. The HSI-setup is validated by measuring series of unfrozen and frozen samples of a hemoglobin-solution, a hemoglobin-intralipid mixture and whole blood with varying oxygen saturation. The theoretically predicted linear relation between oxygen saturation and absorbance was observed in both the frozen sample series and the unfrozen series. In a final proof of principal, frozen myocardial tissue was measured. Higher saturation values were recorded for ventricle and atria tissue compared to the septum and connective tissue. These results are not validated by measurements with another method. The formation of methemoglobin during freezing and the presence of myoglobin in the tissue turned out to be possible sources of error.

The increasing need for highly polychromatic approaches to flow cytometry, coupled with rapid technological advances, have driven the design and implementation of commercial instruments that measure up to 19 parameters using multiple lasers for excitation, an intricate optical filter/mirror arrangement, and analysis using fluorescence compensation approaches. Although such conventional multiparameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from higher density of spectral information and a more flexible approach to spectral analysis including, but certainly not limited to: spectral deconvolution of overlapping spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, cellular autofluorescence characterization, and flow based Raman spectroscopy. For these purposes, we have developed a high resolution spectral flow cytometer using an EMCCD camera with 1600 by 200 pixels, which is capable of detecting less than 200 fluorescein molecules with a spectral resolution of less than 3 nm. This instrument will enable high throughput characterization of single cell or particle emission spectra. For proof of principle instrument operation, we have begun characterization of intrinsic cellular autofluorescence, which is the major source of background for cell-based fluorescence assays. Specifically, we will describe recent work on the high resolution spectral characterization of autofluorescence for several commonly used cell types. Autofluorescence emission is known to cover over almost the entire spectrum from 300 to nearly 800 nm. These emissions are attributed to flavins, elastin, Indolamine dimers and trimers, NADH and collagen among other molecules. We will show that several unique autofluorescence spectra arise in the different cell lines thereby suggesting the possibility of discrimination of cell types based on intrinsic fluorescence.

Cellular autofluorescence, though ubiquitous when imaging cells and tissues, is often assumed to be small in comparison to the signal of interest. Uniform estimates of autofluorescence intensity obtained from separate control specimens are commonly employed to correct for autofluorescence. While these may be sufficient for high signal-to-background applications, improvements in detector and probe technologies and introduction of spectral imaging microscopes have increased the sensitivity of fluorescence imaging methods, exposing the possibility of effectively probing the low signal-to-background regime. With spectral imaging, reliable monitoring of signals near or even below the noise levels of the microscope is possible if autofluorescence and background signals can be accurately compensated for. We demonstrate the importance of accurate autofluorescence determination and utility of spectral imaging and multivariate analysis methods using a case study focusing on fluorescence confocal spectral imaging of host-pathogen interactions. In this application fluorescent proteins are produced when bacteria invade host cells. Unfortunately the analyte signal is spectrally overlapped and typically weaker than the cellular autofluorescence. In addition to discussing the advantages of spectral imaging for following pathogen invasion, we present the spectral properties of mouse macrophage autofluorescence. The imaging and analysis methods developed are widely applicable to cell and tissue imaging.

We present the implementation of a fluorescence lifetime imaging microscopy (FLIM) system for cellular characterisation. FLIM system can be used as an investigative tool to identify minor biochemical changes in cellular abnormalities. These subtle changes could possibly alter cellular fluorescence properties such as emission wavelength and lifetime. In this study, the fluorescence lifetime of haematoxylin and eosin (H&E)-stained tissues were investigated using a wide-field time-domain FLIM system. The transient response of epithelial fluorescence was investigated and the lifetime extracted using a bi-exponential model. It was found that the fluorescence lifetimes of eosin can be correlated to the tissue histology. The preliminary result suggests that tumor-associated molecules are retained in the tissues even after tissue fixation and staining. The developed FLIM system was successfully applied to detect the histological changes in the tissue samples. Optimization of system parameters is also discussed.

We report on a novel approach to study cells and tissues exposed to laser radiation. By using a tightly focused laser beam, a selected area of a cell or a tissue can be selectively irradiated, and the results of this interaction can be immediately interrogated using Raman confocal microscopy. We present our experimental results for skin and eye tissues and individual retinal pigmented epithelium cells demonstrating a great potential of this new research paradigm.

DNA methylation plays a key role in cellular differentiation. Aberrant global methylation patterns are associated with several cancer types, as a result of changes in long-term activation status of up to 50% of genes, including oncogenes and tumor-suppressor genes, which are regulated by methylation and demethylation of promoter region CpG dinucleotides (CpG islands). Furthermore, DNA methylation also occurs in nonisland CpG sites (> 95% of the genome), present once per 80 dinucleotides on average. Nuclear DNA methylation increases during the course of cellular differentiation while cancer cells usually show a net loss in methylation. Given the large dynamic range in DNA methylation load, the methylation pattern of a cell can provide a valuable distinction as to its status during differentiation versus the disease state. By applying immunofluorescence, confocal microscopy and 3D image analysis we assessed the potential of differential nuclear distribution of methylated DNA to be utilized as a biomarker to characterize cells during development and when diseased. There are two major fields that may immediately benefit from this development: (1) the search for factors that contribute to pluripotency and cell fate in human embryonic stem cell expansion and differentiation, and (2) the characterization of tumor cells with regard to their heterogeneity in molecular composition and behavior. We performed topological analysis of the distribution of methylated CpG-sites (MeC) versus heterochromatin. This innovative approach revealed significant differences in colocalization patterns of MeC and heterochromatin-derived signals between undifferentiated and differentiated human embryonic stem cells, as well as untreated AtT20 mouse pituitary tumor cells compared to a subpopulation of these cells treated with 5-azacytidine for 48 hours.

We describe a scanning fluorescence lifetime imaging (SFLIM) system that provides a large field of view (LFOV), using a femtosecond (fs) pulsed laser, for multi-mode optical imaging of small animals. Fluorescence lifetime imaging (FLIM) can be a useful optical method to distinguish between fluorophores inside small animals. However, difficulty arises when LFOV is required in FLIM using a fs pulsed laser for the excitation of the fluorophores at low wavelengths (<500nm), primarily because the field of view of the pulsed blue excitation light generated from the second harmonic of the fs pulsed light is limited to about a centimeter in diameter due to the severe scattering and absorption of the light inside tissues. Here, we choose a scanning method in order to acquire a FLIM image with LFOV as one alternative. In the SFLIM system, we used a conventional cooled CCD camera coupled to an ultra-fast time-gated intensifier, a tunable femtosecond laser for the excitation of fluorophores, and an x-y moving stage for scanning. Images acquired through scanning were combined into a single image and then this reconstructed image was compared with images obtained by spectral imaging. The resulting SFLIM system is promising as an alternative method for the FLIM imaging of small animals, containing fluorophores exited by blue light, for LFOV applications such as whole animal imaging.

Cytomics is the high-content analysis of cell-systems [6, 78]. The area of Cytomics and Systems Biology received great attention during the last years as it harbours the promise to substantially impact on various fields of biomedicine, drug discovery, predictive medicine [6] and may have major potential for regenerative medicine. In regenerative medicine Cytomics includes process control of cell preparation and culturing using non-invasive detection techniques, quality control and standardization for GMP and GLP conformity and even prediction of cell fate based on sophisticated data analysis. Cytomics requires quantitative and stoichiometric single cell analysis. In some areas the leading cytometric techniques represent the cutting edge today. Many different applications/variations of multicolour staining were developed for flow- or slide-based cytometry (SBC) analysis of suspensions and sections to whole animal analysis [78]. SBC has become an important analytical technology in drug discovery, diagnosis and research and is an emerging technology for systems analysis [78]. It enables high-content high-throughput measurement of cell suspensions, cell cultures and tissues. In the last years various commercial SBC instruments were launched principally enabling to perform similar tasks. Standardisation as well as comparability of different instruments is a major challenge. Hyperspectral optical imaging may be implemented in SBC analysis for label free cell detection based on cellular autofluorescence [3]. All of these developments push the systemic approach of the analysis of biological specimens to enhance the outcome of regenerative medicine.

Commercial flow cytometers use photomultiplier tubes (PMTs) for fluorescence detection. These detectors have high linear gain and broad dynamic range, but have limited sensitivity in the red and near infrared spectral regions. We present a comparison of avalanche photodiodes (APDs) and PMTs as detectors in flow cytometry instruments, and demonstrate improved sensitivity and resolution in the red and near infrared spectral regions using the APD. The relative performance of the PMT and APD were evaluated by simultaneously measuring the mean fluorescence intensity and coefficient of variation for emission from light emitting diode pulses, flow cytometry test beads, and fluorescently labeled cells. The relative signal to noise performance of the APD and PMT was evaluated over the 500 nm to 1050 nm wavelength range using pulsed light emitting diode light sources. While APDs have higher quantum efficiency but lower internal gain than PMTs, with appropriate external amplification the APD has signal to noise response that is comparable to PMTs in the 500 nm to 650 nm range and improved response in the 650 nm to 850 nm range The data demonstrates that the APD had performance comparable to the PMT in the spectral region between 500 to 650 nm and improved performance in the range of 650 to 1000 nm, where the PMT performance is quite poor. CD4 positive lymphocyte populations were easily identified in normal human blood both by APD and PMT using phycoerythrin labeled antibodies. In contrast, only the APD detector could resolve CD4 positive populations using 800 nm Quantum dot labeled antibodies.

Complex immunophenotyping single-cell analysis are essential for systems biology and cytomics. The application of cytomics in immunology and cardiac research and diagnostics is very broad, ranging from the better understanding of the cardiovascular cell biology to the identification of heart function and immune consequences after surgery. TCPC or Fontan-type circulation is an accepted palliative surgery for patients with a functionally univentricular heart. Protein-losing enteropathy (PLE), the enteric loss of proteins, is a potential late complication after TCPC surgery. PLE etiology is poorly understood, but immunological factors seem to play a role. This study was aimed to gain insight into immune phenotype alterations following post-TCPC PLE. Patients were studied during routine follow-up up to 5yrs after surgery, blood samples of TCPC patients without (n=21, age 6.8±2.6 years at surgery; mean±SD) and with manifest PLE (n=12, age 12.8± 4.5 years at sampling) and age matched healthy children (control, n=22, age 8.6±2.5 years) were collected. Routine laboratory, immune phenotype and serological parameters were determined. Following PLE the immune phenotype dramatically changed with signs of acute inflammation (increased neutrophil and monocyte count, CRP, IL-8). In contrast, lymphocyte count (NK-cells, αβTCR⁺CD4⁺, αβTCR⁺CD8⁺ cells) decreased (p<0.001). The residual T-cells had elevated CD25 and CD69 expression. In PLE-patients unique cell populations with CD3⁺αβ/γδTCR^- and αβTCR⁺CD4^-8^- phenotype were present in increased frequencies. Our studies show dramatically altered leukocyte phenotype after PLE in TCPC patients. These alterations resemble to changes in autoimmune diseases. We conclude that autoimmune processes may play a role in etiology and pathophysiology of PLE.

Flow cytometric detection of specific rare-event targets within high-background samples such as water or food are frequently defeated by the extremely large population of non-target background particles. Time-gated detection of long lifetime fluorescence (>10μs) labeled microbial targets has been proven highly efficient in suppressing this non-target autofluorescent (<0.1μs) background. A time-gated luminescence (TGL) flow cytometer using UV LED excitation has demonstrated the successful detection of rare-event particles in high autofluorescence background samples. In this report, high-quality 5μm europium beads were made (homogenous intensity and aggregation free) for a detailed evaluation of the prototype performance. The known number of beads (10±2, 100±20 and 1000±100) were first sorted by a conventional flow cytometry sorter, and spiked into an environmental water concentrate (1 ml; containing >10 million non-target particles). The recovery rate for counting these very-rare-event particles using the TGL flow cytometer was then found to be 100%±20% between bead concentrations evaluated.

Introduction: The International Society for Analytical Cytology, ISAC, is developing a new combined flow and image Analytical Cytometry Standard (ACS). This standard needs to serve both the research and clinical communities. The clinical medicine and clinical research communities have a need to exchange information with hospital and other clinical information systems. Methods: 1) Prototype the standard by creating CytometryML and a RAW format for binary data. 2) Join the ISAC Data Standards Task Force. 3) Create essential project documentation. 4) Cooperate with other groups by assisting in the preparation of the DICOM Supplement 122: Specimen Module and Pathology Service-Object Pair Classes. Results: CytometryML has been created and serves as a prototype and source of experience for the following: the Analytical Cytometry Standard (ACS) 1.0, the ACS container, Minimum Information about a Flow Cytometry Experiment (MIFlowCyt), and Requirements for a Data File Standard Format to Describe Flow Cytometry and Related Analytical Cytology Data. These requirements provide a means to judge the appropriateness of design elements and to develop tests for the final ACS. The requirements include providing the information required for understanding and reproducing a cytometry experiment or clinical measurement, and for a single standard for both flow and digital microscopic cytometry. Schemas proposed by other members of the ISAC Data Standards Task Force (e.g, Gating-ML) have been independently validated and have been integrated with CytometryML. The use of netCDF as an element of the ACS container has been proposed by others and a suggested method of its use is proposed.

Autofluorescence emission is commonly measured in flow cytometry and is used as a negative control in protocols that explore binding of exogenous fluorophores to cell receptors or other targets of interest. The presence of intrinsic fluorophores however may burden complex cytometry applications. For example it may be difficult to resolve fluorescence signals from multi-intensity and multi-color measurements when the de-convolved fluorescence in question falls close to the autofluorescence background. One possible solution to intensity and spectral overlap problems in flow cytometry is to acquire fluorescence decay kinetic measurements. To this end we focus on advancing time-dependent flow cytometry and conduct measurements of endogenous fluorescence lifetime. Instrument developments to a phase-sensitive flow cytometry (PSFC) system were coupled with lifetime measurements of intrinsic fluorophores from viable cell samples. The average lifetime of >300,000 individual rat fibroblast cells was measured at discrete wavelengths ranging from 457- to 785-nm using a 10-MHz intensity-modulated excitation beam. AC amplitude, DC, and phase-shift were resolved and the average lifetime from excitable endogenous species was measured. The lifetime results ranged from 1- to 6-ns over the broad spectral range. Cataloging lifetime values prefaces the use of phase-sensitive techniques in more complex systems and provides a priori measurements necessary for PSFC filtering known lifetime signals from Raman, or other emission and scattering events.

Filamentous fungi like Neurospora crassa are a large and evolutionary successful group of organisms that can efficiently colonise microconfined networks like soil, wood, leaf litter, plant and animal tissues. Growth of the fungus Neurospora crassa was monitored for three-dimensional interactions with artificial profiled surface and two-dimensional interactions with a patterned surface. Specific growth parameters that included branching angles and branching distances were used to measure the responses of growing hyphae to the confining features. In three dimensional microfluidic and mazelike structures, changes in the growth parameters were observed and revealed an exceptional directional memory by growing hyphae that was maintained over long distances. Comparison with data from a previous study using another species revealed that different fungal species exhibit surprisingly specific sets of growth parameters. A second set of experiments showed that Neurospora crassa had a distinct affinity to edges and tips. On a surface covered with microscale pyramids, hyphae balanced on and bridged between tips.

To produce a large increase in total throughput, a multi-stage microfluidics system (US Patent pending) is being developed for flow cytometry and closed system cell sorting. The multi-stage system provides for sorting and re-sorting of cohorts of cells beginning with multiple cells per sorting unit in the initial stages of the microfluidic device and achieving single cell sorting at subsequent stages. This design theoretically promises increases of 2- or 3-orders of magnitude in total cell throughput needed for cytomics applications involving gene chip or proteomics analyses of sorted cell subpopulations. Briefly, silicon wafers and CAD software were used with SU-8 soft photolithography techniques and used as a mold to create Y-shaped, multi-stage microfluidic PDMS chips. PDMS microfluidic chips were fabricated and tested using fluorescent microspheres driven through the chip by a microprocessor-controlled syringe drive and excited on an inverted Nikon fluorescence microscope. Inter-particle spacings were measured and used as experimental data for queuing theory models of multi-stage system performance. A miniaturized electronics system is being developed for a small portable instrument. A variety of LED light sources, waveguides, and APD detectors are being tested to find optimal combinations for creating an LED-APD configuration at the entry points of the Y-junctions for the multi-stage optical PDMS microfluidic chips. The LEDs, APDs, and PDMS chips are being combined into an inexpensive, small portable, closed system sorter suitable for operation inside a standard biohazard hood for both sterility and closed system cell sorting as an alternative to large, expensive, and conventional droplet-based cell sorters.

This paper reports a complete on-chip high resolution lensless imaging device based on the optofluidic microscopy method, which can form a vital optical microscopy component in a wide range of lab-on-a-chip systems. This imaging device does not use any lens elements and yet is capable of resolution comparable to that of a conventional microscope with a 20× objective. We demonstrate the use of the device for Caenorhabditis elegans and microsphere imaging at a resolution of ~ 1 μm with an imaging time of ~2 sec. The fabrication of this on-chip imaging device is fully compatible with existing semiconductor and microfluidic technologies, so the device can be massively fabricated and integrated into microsystems to form compact and low-cost total analysis systems for biological and colloidal studies.

Optical trapping by use of multiple counter-propagating beam traps has not been widely implemented outside optical engineering laboratories. One, if not the primary, reason for this is the relatively complex calibration procedures involved in connection with this optical geometry. In this talk, we present automated solutions to all the calibration issues, which in effect results in a turn-key counter-propagating multi-beam 3D trapping system. These results allow a wider audience to utilize counter-propagating beam trapping systems. The calibrated system can be used to independently manipulate a plurality of cells real-time in a large 3D working area. Optionally, the system can be extended to allow for use of various spectroscopic methods concurrently with optical manipulation/trapping.

Many in vitro studies require a pure clonal population of cells that derive from a single cell. Traditionally this task has been performed using the inefficient manual process of ultimate limiting dilution. We have developed a novel clonal dilution technique using the Laser Enabled Analysis and Processing (LEAP^TM) instrument (Cyntellect Inc. San Diego, CA). The LEAP instrument performs automated fluorescence imaging and real time image analysis to identify and measure fluorescence and morphological parameters of cells. The LEAP instrument also features a laser that can be used to manipulate targeted cells. To perform clonal dilution, cells are seeded at a low density (~10 cells/well) into each well of a 384 well plate and viably stained. The LEAP instrument will then image each well and automatically target all of the cells that are present. Then one cell will be chosen to keep (at random or based on a variety of metrics) and the others will be eliminated by laser ablation. We have successfully used this technique to produce single cell clones of HCT116 cells, a heterogeneous colorectal cancer model, in 84 percent of wells (originally containing 5 ± 2.1 cells/well). This is a marked improvement over the traditional technique of ultimate limiting dilution which produces a clone in only 33 percent of wells. The ability to efficiently produce clonal colonies has great utility in the isolation of subpopulations of cancer cells and purification of transformed cell lines.

Because of the softness of membrane, erythrocytes (red blood cell, RBC) have different shapes while being immersed in buffer with different osmotic pressure. While affecting by different viruses and illnesses, RBC may change its shape, or its membrane may become rigid. Moreover, RBC will ford and stretch when it is trapped by optical tweezers. Therefore, the behaviors of RBC in optical tweezers raise more discussion. In this report, we set up an optical tweezers to trap RBC of small animals like feline and canine. By adding a long working distance objective to collect the side-viewing image, a 3-D image system was constructed to detect the motion of trapped RBC. To improve the image quality for side-view, an aperture and narrow glass plate were used. From the video of these images and their spatial spectrum, the shape of trapped RBC was studied.

The migration induced by intensive light is termed photophoresis. We could show that the evaluation of light-induced velocities of microparticles, bacteria and cells suspended in water is valuable for the prediction of their intrinsic properties. Two different laser setups were evaluated for photophoretic migration, a He-Ne laser (P = 45 mW, λ = 633 nm) and a diode-pumped cw-Nd:YAG (P = 1.1 W, λ = 532 nm). When analyzing the migration behavior of particles, we find significant differences depending on both, geometrical size and refractive index. We describe migration of PS particles of different size as well as with different refractive index but same diameter, SiO₂ and melamine resin. The potential for the separation of biological matter is shown as velocity distributions of heat killed bacteria of Escherichia coli, Salmonella enteritidis, and baker's yeast is reported.

Recent semiconductor technology has reduced the size of a laser to the size of a biological cell or even a virus particle. By integrating these ultra small lasers with biological systems, it is possible to create micro-electrical mechanical systems (MEMS) devices that are rapidly finding new applications for chemical analysis, molecular detection, and health care.1-5 One is a nanolaser device that confines intense light into an extremely small interaction volume.6-10 The nanolaser has been integrated with a microfluidic chip and applied to assess novel biomaterials, cells, and organelles. Importantly, these biomaterials can be analyzed without time delays or difficulties associated with chemical fixing or fluorescent markers. With these advantages, nanolaser spectroscopy represents a powerful tool for the rapid analysis of bioparticles such as cells, organelles, vesicles, virions, and other bioparticles.

Based upon a collection of compact LEDs (light-emitting diodes) and a compact photodiode, we have developed a calibration tool for fluorescence microscopes that are used as digital imaging devices. The entire device (excluding a USB connector) measures 25 mm × 80 mm × 12 mm. Virtually all commonly-used fluorophores can be simulated with one of the six LEDs. An LED is chosen from the host computer and its current range is selected (digitally) so as to provide a test of the complete dynamic range of the imaging system. Thus by varying the current through an LED in a controlled way, a controlled amount of "emission" light can be produced, transmitted through the chosen optical path of the microscope, and measured by the image sensor. The digitized intensity can then be determined as a function of the LED current. Any other (fluorescence) intensity measured through the same electro-optical path can then be characterized (and thus calibrated) by an equivalent electrical current. The excitation light is calibrated by a photodiode which has a dynamic range of 10^5:1 and thus is suitable for a variety of light sources: mercury lamps, lasers, LEDs, etc. The integration time of the photodiode as well as its gain can be digitally selected from the host computer. Further, using a Spectralon® reflector, the inherent non-linearity of the LED emission versus current can be measured by the photodiode and used to provide a look-up table compensation independent of the image sensor used in the fluorescence microscope system.

We demonstrate a novel method of two-dimensional differential interference contrast (DIC) microscopy. Our method is cheaper, more compact, and more robust compared to conventional DIC microscopes; since it uses a simple variation of Young's double-slit geometry, no expensive or complex optical components are needed. In addition, our method quantitatively measures differential phase, unlike conventional DIC, which makes our device useful for optical metrology and cell biology applications. The device consists of four circular holes arranged in a "plus" pattern, milled into a metal layer 80 μm above a complimentary metal-oxide semiconductor (CMOS) image sensor. Light incident upon the four-hole aperture is transmitted through the holes and creates an interference pattern on the CMOS sensor. This pattern shifts as a function of the spatial phase gradient of the incident light. By capturing the amplitude and location of the zero-order fringe of the interference pattern, the amplitude and differential phase of the incident light can be measured simultaneously. In this article, we model the response of the device using both geometric optics and Huygens principle. We then verify these models by experimentally measuring the responsivity of our device. A short analysis on the algorithm used to calculate the fringe location follows. We then show a beam profiling application by measuring the amplitude and spatial phase gradient of a Gaussian laser beam and an optical vortex. Finally, we show a DIC microscope application; we image a phase mask of the letters "CIT".

The reliability of lanthanide luminescence measurements, by both flow cytometry and digital microscopy, will be enhanced by the availability of narrow-band emitting lanthanide calibration beads. These beads can also be used to characterize spectrographic instruments, including microscopes. Methods: 0.5, 3, and 5 micron (µm) beads containing a luminescent europium-complex were manufactured and the luminescence distribution of the 5 µm beads was measured with a time-delayed luminescence flow cytometer and a timedelayed digital microscope. The distribution of the luminescence intensity from the europium-complex in individual beads was determined on optical sections by confocal microscopy. The emission spectra of the beads under UV excitation were determined with a PARISS® spectrophotometer. The kinetics of the luminescence bleaching caused by UV irradiation were measured under LED excitation with a fluorescence microscope. Results: The kinetics of UV bleaching were very similar for the 0.5, 3, and 5 µm beads. Emission peaks were found at 592, 616, and 685 nanometers (nm). The width of the principal peak at half-maximum (616 nm) was 9.9 nm. The luminescence lifetimes in water and in air were 340 and 460 microseconds (µs), respectively. The distribution of the europium- complex in the beads was homogeneous. Conclusions: The 5 µm beads can be used for spectral calibration of microscopes equipped with a spectrograph, as test particles for time-delayed luminescence flow cytometers, and possibly as labels for macromolecules and cells.

An automated high-content screening microscope has been developed which uses fluorescence anisotropy imaging and fluorescence lifetime microscopy to identify Förster resonant energy transfer between eGFP and mRPF1 in drug screening assays. A wide-field polarization resolved imager is used to simultaneously capture the parallel and perpendicular components of both eGFP and mRFP1 fluorescence emission to provide a high-speed measurement of acceptor depolarization. Donor excited state lifetime measurements performed using laser scanning microscopy is then used to determine the FRET efficiency in a particular assay. A proof-of-principle assay is performed using mutant Jurkat human T-cells to illustrate the process by which FRET is first identified and then quantified by our high-content screening system.

Fluorescence techniques are known for their high sensitivity and are widely used as analytical tools, detection methods and imaging applications for product and process control, material sciences, environmental and bio-technical analysis, molecular genetics, cell biology, medical diagnostics, and drug screening. According to DIN/ISO 17025 certified standards are used for steady state fluorescence diagnostics, a method having the drawback of giving relative values for fluorescence intensities only. Therefore reference materials for a quantitative characterization have to be related directly to the materials under investigation. In order to evaluate these figures it is necessary to calculate absolute numbers such as absorption/excitation cross sections and quantum yield. This has been done for different types of dopands in different materials such as glass, glass ceramics, crystals or nano crystalline material embedded in polymer matrices. Samples doped with several fluophores of different emission wavelengths and decay times are required for fluorescent multiplexing applications. Decay times shorter than 100 ns are of special interest. In addition, a proper knowledge is necessary of quantum efficiency in highly scattering media. Recently, quantum efficiency in YAG:Ce glass ceramics has been successfully investigated. Glass and glass ceramics doped with threefold charged rare earth elements are available. However, these samples have the disadvantage of emission decay times much longer than 1 microsecond, due to the excitation and emission of their optical forbidden electronic transitions. Therefore first attempts have been made to produce decay-time standards based on organic and inorganic fluophores. Stable LUMOGEN RED pigments and YAG:Ce phosphors are diluted simultaneously in silicone matrices using a wide range of concentrations between 0.0001 and 2 wt%. Organic LUMOGEN RED has decay times in the lower nanosecond range with a slight dependency on concentration and temperature. In addition, the well-known decay properties of inorganic YAG:Ce are observed also embedded in silicone matrix. Luminescent silicone layers are obtained with thicknesses between 150 and 300 µm and no change of decay time, which has been determined to be between 60 and 62 ns. Finally, first results are shown for fluorescent CaF2:Pb glass ceramics embedded in a silicate glass matrix. Wavelength accuracy and lifetime are characterized for different environmental conditions such as temperature treatment and UV irradiation. Moreover, intensity patterns, e.g. line profiles and results, are discussed on homogeneity and photo and thermal stability, respectively. Fluorescence (steady state, decay time) and absorption (remission, absorption) spectroscopy are employed as diagnostic methods to get a microscopic view of the relevant physical processes. The work is funded by BMBF under project number 13N8849.

A Complementary Metal Oxide Semiconductor (CMOS) camera (1024×1024 pixels) is used to record spontaneous oscillations of hair cell stereocillia in an in-vitro preparation of the bullfrog sacculus with the otolithic membrane removed. The CMOS camera is attached to an Olympus BX51WI Microscope inside of a sound-isolation chamber, with white light transmission illumination using an X-Cite 120 metal halogenide lamp. The combination of the parallel readout of the CMOS chip and the high intensity of illumination allows full frame images of the oscillations to be taken at 1000 frames per second. A weighted, time averaged differential algorithm is used to aid in the visualization of the hair cell movement. To detect the displacement from its center of the stereocillia tip with nanometer position resolution and millisecond time resolution, an average background intensity value was subtracted from each image to remove lamp intensity fluctuations and then a center of intensity algorithm was applied. This combination of our imaging system and data analysis allows for the oscillations of more than one hair cell to be recorded during the same time period, and their frequency components extracted.

Nucleotide genomic signals satisfy regularities that reveal restrictions in the distribution of nucleotides and pairs of nucleotides along DNA sequences. Structurally, a chromosome appears to be more than a plain text, by satisfying symmetry constrains that evoke the rhythm and rhyme in poems. These regularities make it easy to identify exogenous inserts in the genomes of prokaryotes, because such inserts obey different regularities than the background sequence. The paper presents instances of inserts found in the genomes of Bacillus subtilis, Mycobacterium tuberculosis and other prokaryotes. Inserts of exogenous material are frequently accompanied by complementary inserts tending to restore the original constrains.

Background: Fluorescence diagnostics uses the ability of tissues to fluoresce after exposition to a specific wavelength of light. The change in fluorescence between normal and progression to cancer allows to see early cancer and precancerous lesions often missed by white light. Aim: To improve by computer image processing the sensitivity of fluorescence images obtained during examination of skin, oral cavity, vulva and cervix lesions, during endoscopy, cystoscopy and bronchoscopy using Xillix ONCOLIFE. Methods: Function of image f(x,y):R² → R³ was transformed from original color space RGB to space in which vector of 46 values refers to every point labeled by defined xy-coordinates- f(x,y):R² → R⁴⁶. By means of Fisher discriminator vector of attributes of concrete point analalyzed in the image was reduced according to two defined classes defined as pathologic areas (foreground) and healthy areas (background). As a result the highest four fisher's coefficients allowing the greatest separation between points of pathologic (foreground) and healthy (background) areas were chosen. In this way new function f(x,y):R² → R⁴ was created in which point x,y corresponds with vector Y, H, a*, c₂. In the second step using Gaussian Mixtures and Expectation-Maximisation appropriate classificator was constructed. This classificator enables determination of probability that the selected pixel of analyzed image is a pathologically changed point (foreground) or healthy one (background). Obtained map of probability distribution was presented by means of pseudocolors. Results: Image processing techniques improve the sensitivity, quality and sharpness of original fluorescence images. Conclusion: Computer image processing enables better visualization of suspected areas examined by means of fluorescence diagnostics.

One of the most challenging problems in medical imaging is to "see" a tumour embedded into tissue, which is a turbid medium, by using fluorescent probes for tumour labeling. This problem, despite the efforts made during the last years, has not been fully encountered yet, due to the non-linear nature of the inverse problem and the convergence failures of many optimization techniques. This paper describes a robust solution of the inverse problem, based on data fitting and image fine-tuning techniques. As a forward solver the coupled radiative transfer equation and diffusion approximation model is proposed and compromised via a finite element method, enhanced with adaptive multi-grids for faster and more accurate convergence. A database is constructed by application of the forward model on virtual tumours with known geometry, and thus fluorophore distribution, embedded into simulated tissues. The fitting procedure produces the best matching between the real and virtual data, and thus provides the initial estimation of the fluorophore distribution. Using this information, the coupled radiative transfer equation and diffusion approximation model has the required initial values for a computational reasonable and successful convergence during the image fine-tuning application.

We have studied the application of the diffusion mapping technique to dimensionality reduction and clustering in multidimensional optical datasets. The combinational (input-output) data were obtained by sampling search spaces related to optimization of a nonlinear physical process, short-pulse second harmonic generation. The diffusion mapping technique hierarchically reduces the dimensionality of the data set and unifies the statistics of input (the pulse shape) and output (the integral output intensity) parameters. The information content of the emerging clustered pattern can be optimized by modifying the parameters of the mapping procedure. The low-dimensional pattern captures essential features of the nonlinear process, based on a finite sampling set. In particular, the apparently parabolic two-dimensional projection of this pattern exhibits regular evolution with the increase of higher-intensity data in the sampling set. The basic shape of the pattern and the evolution are relatively insensitive to the size of the sampling set, as well as to the details of the mapping procedure. Moreover, the experimental data sets and the sets produced numerically on the basis of a theoretical model are mapped into patterns of remarkable similarity (as quantified by the similarity of the related quadratic-form coefficients). The diffusion mapping method is robust and capable of predicting higher-intensity points from a set of low-intensity points. With these attractive features, diffusion mapping stands poised to become a helpful statistical tool for preprocessing analysis of vast and multidimensional combinational optical datasets.

Multi parameter flow cytometry enables detailed identification of cell type and function based on fluorescence of antibody conjugated dye labels. Current instruments use photomultiplier tube detectors to measure up to eight fluorescent labels from a single excitation source. We demonstrate polychromatic flow cytometry using a 14-element avalanche photodiode (APD) array coupled with a dispersive optical grating. Forward scatter, side scatter, and 14 fluorescence channels over the 530 to 800 nm spectral range are recorded using a 16 channel electronics console for simultaneous event capture. The APD detector elements have a working spectral range from 400 nm to 1050 nm. Results are presented for flow cytometry measurements of Spherotech UltraRainbow test beads, quantum dot labeled polystyrene spheres, and cells with antibody conjugated dye labels. The flow cytometry test bead measurements illustrate the sensitivity and spectral resolution of the APD detector array. The application of the instrument is demonstrated by identifying CD4 positive lymphocyte populations in normal human whole blood samples.

Cancerous tumors are dynamic microenvironments that require unique analytical tools for their study. Better understanding of tumor microenvironments may reveal mechanisms behind tumor progression and generate new strategies for diagnostic marker development, which can be used routinely in histopathological analysis. Previous studies have shown that cell invasion and intravasation are related to metastatic potential and have linked these activities to gene expression patterns seen in migratory and invasive tumor cells in vivo. Existing analytical methods for tumor microenvironments include collection of tumor cells through a catheter needle loaded with a chemical or protein attractant (chemoattractant). This method has some limitations and restrictions, including time constraints of cell collection, long term anesthetization, and in vivo imaging inside the catheter. In this study, a novel implantable device was designed to replace the catheter-based method. The 1.5mm x 0.5mm x 0.24mm device is designed to controllably release chemoattractants for stimulation of tumor cell migration and subsequent cell capture. Devices were fabricated using standard microfabrication techniques and have been shown to mediate controlled release of bovine serum albumin (BSA) and epidermal growth factor (EGF). Optically transparent indium tin oxide (ITO) electrodes have been incorporated into the device for impedance-based measurement of cell density and have been shown to be compatible with in vivo multi-photon imaging of cell migration.

This article aims at the optical parameter reconstruction technology for the frequency- domain measurement of near-infrared diffused light. For mimicking the cervix, a cylindrical model with hole in the middle is used in the simulation and experiments. Concerning the structure of the cervix, Monte-Carlo simulation is adopted for describing the photon migration in tissue and Perturbation Monte-Carlo is used for the reconstruction of the optical properties of cervix. The difficulties in the reconstruction of cervical optical properties with frequency domain measurement are the description of the tissue boundary, expression of the frequency-domain signal, and development of rapid reconstruction method for clinical use. To get the frequency domain signal in Monte Carlos simulation, discrete Fourier transformation of the photon migration history in time-domain is employed. By combining the perturbation Monte-Carlo simulation and the LM optimization technology, a rapid reconstruction algorithm is constructed, by which only one Monte-Carlo simulation is needed. The reconstruction method is validated by simulation and experiments on solid phantom. Simulation results show that the inaccuracy in reconstruction of absorption coefficient is less than 3% for a certain range of optical properties. The algorithm is also proved to be robust to the initial guess of optical properties and noise. Experimental results showed that the absorption coefficient can be reconstructed with inaccuracy of less than 10%. The absorption coefficient reconstruction for one set of measurement data can be fulfilled within one minute.

Propose: To study the optimal concentration and time of incubation of human lung adenocarcinoma cell line (SPC-A-1) labeled with superparamagnetic iron oxide (SPIO) particles in vitro. Methods: Human lung adenocarcinoma cell line (SPC-A-1) was cultured with different concenration of SPIO and different time of incubation (labeled with media containing Fe-PLL: 25μg /mL, 100μg /mL, and 200 μg /mL, and for 30min, 90min, 180min. The phagocytosis of the cells was observed by laser scanning confocal microscopy (LSCM) to determine particle uptake and their distribution in cells. Results: Human lung adenocarcinoma cells(SPC-A-1) have taken up a large amount of SPIO particles within the first 3h. Conclusion: In this study, the concentration of iron with 25μg/ml SPIO and time of incubation for 30min is the optimal condition for labeling the SPC-A-1 with SPIO.

Changes of chlorophyll fluorescence emission spectra of different position leaves were investigated during the later growth stages of rice leaves. Results showed that natural illumination induced a shift change of fluorescence emission intensity (both in P685 and P735) during the progressive senescence of rice leaves. These data suggested that changes of chlorophyll fluorescence emission spectra of different position leaves were possible due to the declined of energy and electron transport activities of PSII reaction center. On the other hand, different position leaves of rice were treated with 3-(3,4 dichlorophenyl)-1,1-dimethyl-urea (DCMU), the results showed that DCMU had a significant effect on Chl fluorescence emission spectra (both in P685 and P735) under room temperature and different position leaves had a different sensibility of DCMU. It is concluded that natural illumination plays an important role in the progressive senescence process of rice leaves.

Research of depth of field (DOF) for capsule endoscope is important for the reason that the shapes of the object plane of the intestine or the stomach are curve surfaces of "<" shape or "c" shape. The depth of field is dependent on following factors: focal length, circle of confusion, aperture, and subject distance. The first three factors are improved for wide view angle in prior paper and determined by the chosen sensor, and it is not going against depth of field. Last factor, subject distance, is the more freedom to enlarge the depth of field. However, depth of field is the range between near depth of field limit and far depth of field limit that are acceptably sharp. The fraction of the depth of field behind the focus is always large then the one in front of the focus distance. The depth of field does change with object distance, and it is increasing as object distance is increasing. But the object distance of the design for capsule endoscope is short. The object distance setting in front of the dorm is more efficient to use the depth of field than the one setting at the dome top. Therefore there is an appropriate design of object distance to make depth of field be used efficiently to inspect curve surface of intestine and stomach. The more vision information of inspect digestive system is get and is compared easily to diagnose patients' condition under wide and efficient range of depth of field.

We monitored cell viability and damage under femtosecond laser irradiation using aser weezers Raman pectroscopy (LTRS) which is becoming a powerful tool for the analysis of biological materials. Femtosecond lasers are more frequently used as a light source for optical tweezers since they enable nonlinear optical phenomena such as two-photon absorption or second harmonic generation trapping. Femtosecond laser optical trapping similar to thee CW laser optical trapping except that optical damage can be easily induced due to extremely high peak power of femtosecond pulses. We monitored the Raman signal changes as a marker for optical damage. We used red blood cell (RBC) as a target sample and first used the CW laser beams to trap the RBC from the bottom of the chamber. After the trapped RBC is moved to a desired depth, we switched the laser mode to mode-locked mode and monitored the Raman signals as a function of the laser irradiation time. It was observed that the Raman shift at 1543 cm^-1 may be a good marker for optical damage both for CW and femtosecond laser trapping.

We have developed an automated multicolour high-throughput multi-colour Fluorescence in-situ Hybridization (FISH) scanning system for examining Non-Small Cell Lung Cancer (NSCLC) 5-10μm thick tissue specimens and analyzing their FISH spot signals at the individual cell level and then as clonal populations using cell-cell architecture (spatial distributions). Using FISH probes targeting genomic areas deemed significant to chemotherapy resistance, we aim to identify clonal subpopulations of cells in tissue samples likely to be resistant to cis-platinum/vinorelbine chemotherapy. The scanning system consists of automatic image acquisition, cell nuclei segmentation, spot counting and measuring the spatial distribution and connectivity of cells with specific genetic profiles across the entire section using architectural tools to provide the scoring system.

Compared to other forms of skin cancer, a malignant melanoma has a high risk of spreading to other parts of the body. Melanoma invasion is a complex process involving changes in cell-extracellular matrix (ECM) interaction and cell-cell interactions. To fully understand the factors which control the invasion process, a human skin model system was reconstructed. HBL (a commercially available cell line) melanoma cells were seeded on a skin model with and without the presence of keratinocytes and/or fibroblasts. After 14 days culture, the skin specimens were fixed, parafin embedded and cut into 7 µm sections. The de-parafinised sections were investigated by synchrotron Fourier transformed infrared (FTIR) microspectroscopy to study skin cell invasion behaviour. The advantage of using FTIR is its ability to obtain the fingerprint information of the invading cells in terms of protein secondary structure in comparison to non-invading cells and the concentration of the enzyme (matrix-metalloproteinase) which digests protein matrix, near the invading cells. With aid of the spectral mapping images, it is possible to pinpoint the cells in non-invasion and invasion area and analyse the respective spectra. It has been observed that the protein bands in cells and matrix shifted between non-invasive and invasive cells in the reconstructed skin model. We hypothesise that by careful analysis of the FTIR data and validation by other models, FTIR studies can reveal information on which type of cells and proteins are involved in melanoma invasion. Thus, it is possible to trace the cell invasion path by mapping the spectra along the interface of cell layer and matrix body by FTIR spectroscopy.

Telomeres play a critical role in the maintenance of chromosomal stability. Telomere erosion, coupled with loss of DNA damage checkpoint function, results in genomic instability that promotes the development of cancer. The critical role of telomere dynamics in cancer has motivated the development of technologies designed to monitor telomere reserves in a highly quantitative and high-throughput manner in humans and model organisms. To this end, we have adapted and modified two established technologies, telomere-FISH and laser scanning cytometry. Specifically, we have produced a number of enhancements to the iCys LSC (CompuCyte) package including software updates, use of 60X dry objectives, and increased spatial resolution by 0.2 um size of stage steps. In addition, the 633 nm HeNe laser was replaced with a 532 nm green diode laser to better match the viewing options. Utilization of telomere-deficient mouse cells with short dysfunctional telomeres and matched telomerase reconstituted cultures demonstrated significantly higher mean integral specific fluorescence values for mTR transfectants relative to empty vector controls: 4.485M vs. 1.362M (p<0.0001). Histograms of average telomere intensities for individual cells were obtained and demonstrated intercellular heterogeneity in telomere lengths. The validation of the approach derives from a strong correlation between iCys LSC values and Southern blotting. This validated method greatly increases our experimental throughput and objectivity.

Large-scale analysis of proteins, which are functional biomolecules, has assumed an important role in the life sciences. Mass spectrometry is one of the techniques used to identify ptoreins. Direct desorption and ionization of proteins from polyacrylamide gel is expected to be a the high-throughput technique in proteomics, which will eliminate several problems such as sample loss, adduct formation, and contamination. In this study, we performed direct ionization of a protein, bovine insulin, in a polyacrylamide gel without matrix addition with a tunable MIR nanosecond pulsed laser. Mass spectra of insulin in an acrylamide gel and an acrylamide solution were recorded in the wavelength range 5.8-5.9 μm and at a wavelength of 6.0 μm, respectively. This wavelength corresponds to the >C=O stretching vibration mode of acrylamide.

Optical scattering spectra obtained in the clinical trials of breast cancer diagnostic system were analyzed for the purpose to detect in the dataflow the segments corresponding to malignant tissues. Minimal invasive probe with optical fibers inside delivers white light from the source and collects the scattering light while being moved through the tissue. The sampling rate is 100 Hz and each record contains the results of measurements of scattered light intensity at 184 fixed wavelength points. Large amount of information acquired in each procedure, fuzziness in criteria of 'cancer' family membership and data noisiness make neural networks to be an attractive tool for analysis of these data. To define the dividing rule between 'cancer' and 'non-cancer' spectral families a three-layer perceptron was applied. In the process of perceptron learning back propagation method was used to minimize the learning error. Regularization was done using the Bayesian approach. The learning sample was formed by the experts. End-to-end probability calculation throughout the procedure dataset showed reliable detection of the 'cancer' segments. Much attention was paid on the spectra of the tissues with high blood content. Often the reason is vessel injury caused by the penetrating optical probe. But also it can be a dense vessel net surrounding the malignant tumor. To make the division into 'cancer' and 'non-cancer' families for the tissues with high blood content a special perceptron was learnt exceptionally on such spectra.

In the clinical trials it was shown, that characteristics of optical scattering and absorption are sensitive to the tissue type and state. In the given report improved optical biopsy system will be presented, clinical trials of which have been conducted in the Regional Oncology Center of Nizhny Novgorod, Russia. During a year more than 160 patients with breast tumors were investigated using this system. Radiation from a xenon lamp through an optical fiber placed inside the probe's needle was delivered into the breast. The radiation scattered from the breast tissue was collected by another fibers also placed in the same needle and its spectrum was measured. Obtained optical data was analyzed to find general optical characteristics of scattered radiation in different types of tissue and revealing the major peculiarities in the spectral scattering coefficients of malignant tumors and their distinctions from benign tumors and healthy tissue. Using different mathematical algorithm the typical template of scattering spectrum was found for benign and malignant type of breast tumor. Then the algorithm of automatic detection of malignant spectra in the data flow was developed. Using this algorithm the datasets of all patients were processed and analyzed and the diagnoses were obtained. The automatic diagnoses were compared with those given by physicians. As a result the indexes of sensitivity and specificity for the optical biopsy diagnostic method were found equal to 96% and 80% correspondingly.