Laser Doppler Imaging (LDI) is a non-invasive optical technique used for assessing the blood flow in biological tissue. This technique allows visualizing 2D distribution of blood flow over an extended area of tissue, e.g. human skin. We have developed a new high-speed instrument for full-field blood flow laser Doppler imaging. The new imager delivers high-resolution flow images every 0.7 to 11 seconds, depending on the number of points in the acquired time-domain signal (32-512 points) and the image resolution (256×256 or 512×512 pixels). This new imaging modality utilizes a digital integrating CMOS image sensor to detect Doppler signals in a plurality of points over the area illuminated by a divergent laser beam of a uniform intensity profile. The integrating property of the detector improves the signal-to-noise ratio of the measurements, which results in high-quality flow images. We made a series of measurements in vitro to test the performance of the system in terms of bandwidth, SNR, etc. Subsequently we give some examples of flow-related images measured on human skin, thus demonstrating the performance of the imager in vivo. The perspectives for future implementations of the imager for clinical and physiological applications will be discussed.

Measurement of blood perfusion has been playing an important role in the field of bioheat transfer and clinical thermotherapy. However, it is not easy to gain thermal induced dynamics of blood perfusion of local tissue. Laser speckle techniques we developed were valid. In this paper, we reviewed our progress on studying thermal induced dynamics of blood perfusion of mice's mesentery with laser speckle techniques which included laser speckle microscopy with CCD camera for isolate vessel and laser speckle imaging for local vessels of mesentery of mice. The principles of the two kinds of laser speckle techniques were introduced briefly. The blood perfusion of normal and tumor mice's mesentery were showed during different constant temperatures (41-45°C) by laser speckle imaging technique. Further more, the dynamics of velocity, diameter and blood perfusion for normal and tumor were compared. The results showed that tumor was more sensitive to heat than normal tissue, which accorded with pathological investigation. Finally, the quantitative relationship among the blood perfusion, the temperature-dependent and the damage-dependent was established. The model could describe successfully thermal induced dynamics of blood perfusion of mesentery for both normal and tumor mice.

There is increasing interest in determining drug diffusion rates in ocular tissues. In this paper we present results of our pilot investigation of depth-resolved monitoring of water diffusion in rabbit cornea using Optical Coherence Tomography (OCT) technique. We monitored changes in the local scattering coefficient of rabbit cornea in vitro as water propagated inside the tissue. Tissue hydration and saturation phases were clearly seen. The water diffusion rate was about 1.48^-5cm/sec. The experimental results suggest that water diffusion rate in cornea can be potentially measured with the OCT system noninvasively with high precision and accuracy.

of biomedical imaging techniques, the optical coherence tomography (OCT), photoacoustic (PA) and time-of-flight (TOF) techniques are among the most researched and, consequently, increasingly broadly applied. This article reviews their basic principles, compares their technical aspects and discusses their ability to determine optical parameters. Also studied is their applicability to non-invasive glucose monitoring in Intralipid^TM phantom measurements, followed by a discussion of some detected problems. Although suffering from a limited probing depth, OCT was found to have the best capacity for detecting changes in the light scattering properties of Intralipid^TM. Photon migration studies with the TOF technique showed changes in pulse amplitude, pulse width and arrival time of the pulse maximum as a function of changes in Intralipid^TM concentration, whereas the sensitivity of the PA technique for detecting changes in scattering was less pronounced. However, each technique showed changes in the registered signals when measuring large glucose concentrations in Intralipid^TM phantoms. Measurement results were also affected by the varying measurement geometries and the photon energies of the light sources.

Modern optical diagnostics for quantitative characterization of polydisperse sprays and other aerosols which contain a wide range of droplet size encounter difficulties in the dense regions due to the multiple scattering of laser radiation with the surrounding droplets. The accuracy and efficiency of optical measurements can only be improved if the radiative transfer within such polydisperse turbid media is understood. A novel Monte Carlo code has been developed for modeling of optical radiation propagation in inhomogeneous polydisperse scattering media with typical drop size ranging from 2 μm to 200 μm in diameter. We show how strong variations of both particle size distribution and particle concentration within a 3D scattering medium can be taken into account via the Monte Carlo approach. A new approximation which reduces ~20 times the computational memory space required to determine the phase function is described. The approximation is verified by considering four log-normal drop size distributions. It is found valid for particle sizes in the range of 10-200 μm with increasing errors, due to additional photons scattered at large angles, as the number of particles below than 10 μm increases. The technique is applied to the simulation of typical planar Mie imaging of a hollow cone spray. Simulated and experimental images are compared and shown to agree well. The code has application in developing and testing new optical diagnostics for complex scattering media such as dense sprays.

Our group at the BC Cancer Research Centre has developed an optical computed-tomography microscope for three-dimensional (3-D) imaging and quantitative analysis of absorption-stained biological samples. The device uses non-ionizing optical radiation for illumination and computed-tomography for 3-D image reconstruction. The optical system consists of a light source, two high-numerical aperture (NA) objective lenses, a sample stage, a CCD detector, and a spatial light modulator that is used for computer-controlled sample illumination. Projections of a specimen are measured at a set of appointed angles within the range of 0 less than or equal to angle less than or equal to 135, which is limited by the NA of the objective. The reconstruction algorithm developed to solve the limited-angle problem employs the reconstruction from a transform-based method as an initial guess for the following iterative reconstruction. 3-D microscopic images of quantitatively absorption-stained cells have been generated with the resolution on the order of 1 micron. Visualization of 3-D tissue structure and architectural and morphological features that can be extracted from the volume to provide pathologists with additional information for diagnostic purposes.

Conventional photoacoustic techniques in composition determination and biomedical diagnose and imaging are based on the optical absorption in target substance or objects from which the photons to be scattered are not concerned. It is obvious that the intensities of scattered lights closely relate to the property of the interrogated substance, therefore measuring the signals produced by them can give rise to more information of the substance. Based on this idea, a novel method entitled scattering photoacoustic (SPA) method is put forward to study weak absorption suspensions with highly scattering. In this method, a near infrared pulse laser irradiates the studied object which contacts with external absorbers, resulting the generation of a few photoacoustic signals; one is produced in the studied object as conventional case, others are in external absorbers which are produced by the scattered photons. All these signals are successively received by a piezoelectric detector with short damping period. Analyzing these signals is capable of determining reduced scattered coefficient and absorption coefficient, as well as acoustic attenuation of studied suspensions. Some measurement results in intralipid and fibre (paper pulp) suspensions are given rise to in the end.

Osteoarthritis is a painful condition, causing restricted mobility in the articular joints. In this paper we present a review of different optical techniques that might be used to clarify the etiology of degeneration of connective joint tissues, such as bone and articular cartilage. Significant correlation (R² = 0.8) between bone mineral density and scattering coefficient of cortical bone tissue are found by using Integrating Sphere Technique. Optical Coherence Tomography and Polarization-Sensitive Optical Coherence Tomography images of cartilage tissue are presented. They were performed as series of angle-dependant measurements for different location along the surface. Method for spatial mapping the birefringence of equine articular cartilage is proposed. Variations in band spacing of birefringence obtained from visually healthy and abnormal cartilage samples are compared. Visible osteoarthritic lesions are characterized by a loss of the regular birefringence bands shown by normal cartilage. We discuss the hypothesis that some of these variations may be due to changes in intrinsic structure of tissue.

A few recent applications of lasers and optical technologies in clinical dermatology have already shown its potential not only to be objective, accurate, rapid, reproducible, non-invasive, non-destructive methods, but also able to study pathophysiology and biophysics of the skin, qualitatively and quantitatively perform evaluation and monitoring of the dynamics of skin lesions in vitro, ex vivo and especially in vivo, that have not been described before in any textbook of dermatology. Here are demonstrated some data from clinical dermatological studies by using laser Doppler flowmetry, reflectance spectrophotometry, infrared microspectroscopy and fiberoptic near-infrared Raman spectroscopy for investigating pathophysiological and pharmacological aspects of some dermatological diseases, as well technical availability and reliability of utilized techniques for selected purposes in the clinic. Such a survey of a number of applications by a variety of laser and optical technologies aims to receive much more attention by a dermatological community to further introduce new potential applications by using this kind of technologies in the clinical settings, and also to expand interdisciplinary knowledge considering presently determined advantages and disadvantages of the probed instrumentation in the hospital.

We report the effects on two-photon excitation microscopy of applying optical clearing agents to human skin tissue samples. We demonstrate that the agents glycerol, propylene glycol and glucose in aqueous solution are all effective in enhancing penetration depth (by up to a factor of 2) and in increasing image contrast (by up to a factor of 90 at 80 μm depth) in 150 μm thick sections. We analysed the dynamics of the clearing process, by developing a simple theoretical model based on the free diffusion of the agent into the tissue. In experiments employing simultaneous two-photon excitation and second harmonic generation microscopy similar contrast was produced. A preliminary measurement of the clearing effect on a bulk skin sample is also presented. All three agents are potentially biocompatible and effective in reducing scattering; hence, in improving light penetration depth and image contrast. As such, they could be suitable for in vivo application in two-photon microscopy, as well as in other techniques performing optical biopsy of human skin tissue.

Novel laser structures are required for the next generation of high bit rate optical transmission systems. The objectives of the MONOPLA 1ST project are the study and design of a monolithic structure capable of short pulse, high bit rate generation. In this paper the recent experimental characterisation results of the fabricated devices along with a study of the possibility their use as millimetre wave generators are presented, along with recent theoretical studies in the development of integrated pulse shapers.

This paper reports the results of our investigation in a phantom imaging experiments with a prototype phase contrast x-ray imaging system. Two types of phantoms, including a standard mammography phantom, and a contrast-detail phantom, were imaged, and the impact of the x-ray focal spot sizes and the magnification ratios were investigated. The images acquired with phase contrast prototype system shows better detectability as compared with the images acquired under conventional attenuation base x-ray imaging conditions.

Traditional local cancer treatment modalities include surgery and radiation, which has the immediate tumor response due to tumor removal or radiation induced cell death. However, such therapeutic approaches usually do not result in eradiation of tumors, particularly when treating metastatic tumors. In fact, local treatment of primary tumors may stimulate the growth and spread of remote metastasis. Commonly used systemic therapies include chemotherapy and immunotherapy, which target the dividing cells or the immune systems. However, in addition to the severe side effects, chemotherapy often suppresses the immune systems, hence lessening the host's ability to fight the disease. Immunotherapy, on the other hand, aims at educating and stimulating immune systems using either general immune enhancements or antigen-oriented specific immune stimulation. However, so far, the traditional immunotherapy has yielded only limited success in treating cancer patients. A different approach is needed. To combine the advantages of both local therapies for acute and targeted treatment responses and the systemic therapies for stimulation of the immune systems, laser immunotherapy was proposed to use selective photothermal therapy as the local treatment modality and the adjuvant-assisted immunotherapy for systemic control. Laser immunotherapy has show positive results in treating metastatic tumors. In this study, we conducted a comparative study using surgery, freeze-thaw immunization and laser immunotherapy in the treatment of metastatic rat mammary tumors. Our results showed that removal of the primary tumors was unsuccessful at changing the course of tumor progression. The tumor cell lysate immunization delayed the emergence of metastases but did not provide immunity against the tumor challenge. Laser immunotherapy, on the other hand, resulted in regression and eradication.

Parts of the works of our group in the past five years on near infrared time-resolved (TR) optical tomography are summarized in this paper. The image reconstruction algorithm is based on Newton Raphson scheme with a datatype R generated from modified Generalized Pulse Spectrum Technique. Firstly, the algorithm is evaluated with simulated data from a 2-D model and the datatype R is compared with other popularly used datatypes. In this second part of the paper, the in vitro and in vivo NIR DOT imaging on a chicken leg and a human forearm, respectively are presented for evaluating both the image reconstruction algorithm and the TR measurement system. The third part of this paper is about the differential pathlength factor of human head while monitoring head activity with NIRS and applying the modified Lambert-Beer law. Benefiting from the TR system, the measured DPF maps of the three import areas of human head are presented in this paper.

Light scattering provides a problem in optical spectroscopy as the relationship between attenuation and absorption in the presence of scattering is non-linear. Three simple methods of reducing the effects of scattering are considered in this paper, namely; polarization subtraction, use of an added absorber and spatial filtering. There is an inevitable trade-off between the accuracy of the measurement and the signal to noise ratio as scattered light is rejected from the detector. It is demonstrated that polarization subtraction provides the optimum performance as it enables the relationship between attenuation and absorption coefficient to become more linear at a higher signal to noise ratio than both use of an added absorber and spatial filtering over a range of scattering coefficients.

The investigation of blood dynamical characteristics in the skin under the action of 40% glucose solution was performed in vivo by the laser Doppler technique. Experiments demonstrate that glucose solution affects significantly the blood perfusion and concentration. Qualitative explanation was made for observed perfusion dynamic effect in the skin dermis based on the following factors: tissue cells shrinkage and additional capillaries opening under osmotic stress. The size of glucose vesicle lens was measured under the skin by ultrasonography. The analysis of vesicle sizes monitoring leads to the conclusion that glucose lens spread, basically, along the skin than in the perpendicular to the skin surface direction. Obtained results show the significant anisotropic perturbation of the dynamic characteristics of blood in vascular plexus under the optical active solution influence that must be taken into consideration during optical clarification of biological tissues.

Apoptosis, or programmed cell death, is a form of cell death occurring during normal physiological processes and is used by the multicellular organism for elimination of "old" and impaired cells. Apoptosis is characterized by specific morphological changes such as plasma membrane blebbing, nucleus condensation, and cell wrinkling with further destruction into apoptotic bodies. Apoptosis detection is in focus of instrumental methods used in modern biomedical sciences. The available methods for such purpose are either very expensive, or require time-consuming operations. Their specificity and sensitivity are frequently not sufficient for biomedical diagnostics. We propose to use light scattering analysis for evaluation of apoptosis in cell population, especially for the detection of physical changes of cells, such as cell condensation and degradation into apoptotic bodies. The method proved to be very effective, providing quantitative estimation and high precision, simplicity and low costs of analysis (UA Patent No.64090). Another approach for the detection of apoptosis is based on the recently discovered fact (Bilyy, Stoika 2003; Bilyy et al, 2004; 2005) that apoptotic cells are characterized by increased expression levels of specific glycoproteins in the plasma membrane, which were proved to be selective and specific markers of apoptotic cells. Specific carbohydrate-binding proteins - lectins - were used for identification of mentioned glycoproteins; fluorescent conjugates of lectins were proved to be another novel tool for apoptosis identification using approaches of biophotonics.

NIR tomography has advanced considerably over the past decade. The historical developments which have led to creation of NIR tomography have largely resulted in systems which were optimized for signal accuracy and high numbers of point measurements, rather than speed of acquisition. One of the key technological designs limiting the acquisition speed is the requirement that the source light be delivered sequentially or through frequency encoding of the time signal. The approach presented here provides a method to introduce all sources and all detectors simultaneously with no moving parts and retaining the dynamic range of the detector, by separating the signals spatially prior to detection. This is achieved by spectral wavelength encoding of the sources, which allows many sources to be input into the tissue at the same time, followed by spectral-decoding of all detection channels in parallel using a spectrometer and CCD detector. A data acquisition speed of 35Hz frame rate has been achieved with this configuration. The described system features 8 sources at an overall 785nm center band and 8 detectors evenly deployed in a 27mm array designed for imaging with small animal tissues. This paper discusses the design, instrumentation of the system, and the imaging characteristics.

The study of second harmonic generation (SHG) has been examined using a vectorial approach for both linearly and radially polarized beams. This approach is necessary for situations when the beam is tightly focused such as in a microscope. Using the vectorial approach, the result of including the y and z components of the electric field is that previously ignored 'cross-component' terms are now found to have an influence on the SHG polarization and the radiation patterns obtained. Since SHG is dependent on the susceptibility tensor, the inclusion of these 'cross-component' terms can help to identify structural changes in biological materials simply by studying the changes in the tensor via the SHG polarization. In particular, we calculate the second harmonic polarization induced in collagen for both linearly and radially polarized beams.

The FDTD modeling technique is applied to provide additional insight on the effect of the cell membrane thickness in optical immersion enhanced phase contrast microscope imaging. Pilot results on the application of the FDTD approach for studying the implementation of the optical immersion technique for the visualization of single and multiple gold nanoparticles in biological cells are also presented. The paper focuses on three different scenarios considering single biological cells containing i) cytoplasm and membrane, ii) cytoplasm, nucleus and membrane, and iii) cytoplasm, nucleus, gold nanoparticle(s) and membrane. To the best knowledge of authors, this is the first research study discussing the cell membrane thickness and single gold nanoparticle effects on the forward scattered light from biological cells. The potential of the FDTD approach and its applicability to new and promising biomedical optics research areas such as the study of optical immersion technique enhanced bio-imaging is demonstrated.

From an optical point of view, turbid media like waste water and water in aquacultures are strongly scattering media. The measuring method presented is based on the determination of the optical parameters μ_s (scattering coefficient), μ_a (absorption coefficient) and the anisotropy factor (g), which had originally been developed for the so-called tissue optics in medicine. Double integrating spheres were redesigned for mobile monitoring of optical parameters. A prototype was tested for measuring the change of the absorption of yeast cells with concentration.

Imaging of the human upper airway is widely used in medicine, in both clinical practice and research. Common imaging modalities include video endoscopy, X-ray CT, and MRI. However, no current modality is both quantitative and safe to use for extended periods of time. Such a capability would be particularly valuable for sleep research, which is inherently reliant on long observation sessions. We have developed an instrument capable of quantitative imaging of the human upper airway, based on endoscopic optical coherence tomography. There are no dose limits for optical techniques, and the minimally invasive imaging probe is safe for use in overnight studies. We report on the design of the instrument and its use in preliminary clinical studies, and we present results from a range of initial experiments. The experiments show that the instrument is capable of imaging during sleep, and that it can record dynamic changes in airway size and shape. This information is useful for research into sleep disorders, and potentially for clinical diagnosis and therapies.

In this study Fiber Optic Backscatter Spectroscopic Sensor (FOBSS) is used to monitor the demineralization and remineralization induced changes in the enamel. Application of visible light spectroscopy offers different advantages such as rapid sensing, high spatial resolution, sensitivity, simultaneous examination of wide wavelengths, and permits the application of fiber optic probes. A bifurcated fiber optic bundle probe connected to a visible light source and a high resolution spectrophotometer was used to acquire the backscattered light from the specimen surface. These experiments showed that demineralization and remineralization processes induced a linear decrease in the backscatter light intensity and linear increase in the backscatter light intensity respectively. These experiments highlight the potential advantage of using fiber optic backscatter spectroscopy to determine early enamel demineralization and remineralization.

Influence of glucose addition to specimens of diluted blood on the transmission spectrums have been investigated by experimentally and by the simulation. The coefficients of extinction, scattering and absorption of erythrocytes were calculated with using Mie theory for spherical particles of equal volume. Transmission spectrums were measured with the spectrophotometer Cary500 in the wavelength range 460-860 nm. Specimens of liquid for imbedding of erythrocytes were preparing by mixing blood plasma and 50-% glucose solution with the different concentrations. The volume concentrations (hematocrit) of red blood cells (RBC) were maintained to have the same values in all specimens by adding equal volume of whole blood to immersion liquid of equal volumes. It has been shown that transmission is decreasing for all wavelengths with the addition of glucose solution on condition that the volume concentrations less than 0.35-0.4. The subsequent increase of the glucose concentration results in increasing of spectral transmission.

We designed a dual-sensor instrument for measuring optical signals from the arms of human volunteers. The instrument had two temperature-controlled localized reflectance optical probes. Each probe had one illumination fiber and four detection fibers at different source-detector distances. The two probes were maintained at 30 °C. Thirty seconds after contact with the skin one was heated and the other was cooled at the same rate. The effect of heating and cooling on the signal was measured and correlated with blood glucose concentration. The measurements were performed 3 to 5 times a day for each volunteer over the span of three weeks. The data points from the first two weeks were used to establish a calibration model for each volunteer, which was used to predict glucose values from the third week optical data. Successftil calibration was possible for two of the three volunteers.

We use our new combined functional near infrared spectro-imaging (fNIRSI) and magnetic resonance imaging (MRI) technique to compare fMRI and fNIRSI data at different activation conditions, to obtain new information about the underlying physiology of the blood oxygen level dependent (BOLD) signal used in fMRI, and to assess statistical characteristics of spatial functional information provided by the group analysis of fNIRSI data. To achieve these goals we have acquired simultaneously fNIRSI and fMRJ data during the presentation of the checkerboard reversing with different frequencies, and analyzed these data following the standard correlation and group analysis of variance pathway used in functional neuroimaging. We have found that while the time courses of oxy-, deoxy-, and total- hemoglobin responses are equally well correlated with the time course of the BOLD response, the spatial pattern and magnitude of the BOLD response is better related to those of the oxy-, and total- hemoglobin responses rather than to the deoxyhemoglobin response. The statistical significance of the fNIRSI group maps is inferior to that of fMRI, and can be particularly compromised by the anatomical features of subjects.

Since the advent of the optical microscope around 400 years ago there has been an increasing desire by life science researchers to image ever more deeply into samples with high resolution. More recently this desire has lead to the requirement to image three dimensional, living samples with sub-cellular resolution. The application of practical confocal microscopes partially solved this challenge but it was not until the development of multiphoton imaging methods 15 years ago that true in vivo, imaging with high resolution could take place at depth within samples. This paper reviews the basic principles behind multiphoton microscopy and the advances that have been made in the last five years with regard to real time, in depth, imaging. Consideration is given to the best design of multiphoton instruments along with recent research that has been undertaken in the use of active optical elements to enhance in vivo imaging. Some guidelines for the correct selection of the microscopy method for a range of life science challenges are also presented.

A study is carried out to better understand the spatial properties of light scattered from a turbid medium. The investigation is done within the context of a reflectance mode confocal microscope based on a 4-f optical system. A Monte Carlo simulation to describe the focusing and propagation of light in a turbid medium is adopted. The spatial distribution and phase space profile of the photons on different strategic planes within the system are observed and discussed. Scattering information such as scattering coefficient and the number of scattering events are matched with some of the simulation parameters to produce important information and hence their implications.

In this contribution we show application of multispectral confocal laser scanning microscopy for visualisation and characterisation of polyelectrolyte microcapsules. Fluorescent labels of different free charges, specifically bound by electrostatic interactions to the polymer chains in polyelectrolyte microcapsules, were used for questioning the spatial distribution of polymer components of the microcapsules. To separate the overlapping emission spectra of the fluorescent labels spectral imaging and linear unmixing approaches was used.

For in vivo determination of optically active (chiral) substances in turbid media, like for example glucose in human tissue, the backscattering geometry is particularly convenient. However, recent polarimetric measurements performed in the backscattering geometry have shown that, in this geometry, the relatively small rotation of the polarization vector arising due to the optical activity of the medium is totally swamped by the much larger changes in the orientation angle of the polarization vector due to scattering. We show that the change in the orientation angle of the polarization vector arises due to the combined effect of linear diattenuation and linear retardance of light scattered at large angles and can be decoupled from the pure optical rotation component using polar decomposition of Mueller matrix. For this purpose, the method developed earlier for polar decomposition of Mueller matrix was extended to incorporate optical rotation in the medium. The validity of this approach for accurate determination of the degree of optical rotation using the Mueller matrix measured from the medium in both forward and backscattering geometry was tested by conducting studies on chiral turbid samples prepared using known concentration of scatterers and glucose molecules.

The signal from a confocal measurement as the focal volume is scanned down into a tissue yields an exponential decay versus depth Z_focus, signal = ρ exp(-μ z_focus), where ρ [dimensionless] is the local reflectivity and μ [1/cm] is an attenuation coefficient. A simple theory for how p and μ depend on the optical properties of scattering (μ_s) and anisotropy (g) is presented. Experimental measurements on 5 tissue types from mice (white and gray matter of brain, skin, liver, muscle) as well as 0.1-μm-dia. polystyrene microspheres are presented. The tissues have similar μ_s values (about 500 [1/cm]) but variable g values (0.8-0.99). Anisotropy appears to be the primary mechanism of contrast for confocal measurements such as reflectance-mode confocal laser scanning microscopy (rCLSM) and optical coherence tomography (OCT). While fluorescence imaging depends on fluorophores, and absorption imaging depends on chromophores, the results of this study suggest that contrast of confocal imaging of biological tissues depends primarily on anisotropy.

We determined the scattering coefficient and scattering anisotropy of blood samples with varying hematocrit using optical coherence tomography measurements and a curve fitting procedure. Initial results show much lower scattering coefficient and scattering anisotropy than theoretically expected, which are likely attributed to the highly forward scattering nature of blood.

Aminolevulinic acid (ALA) is an efficient substance used in photodynamic therapy (PDT). It is a precursor of light-sensitive products that can selectively accumulate in malignant cells following the altered activity of the heme biosynthetic pathway enzymes in such cells. These products are synthesized in mitochondria and distributed to various cellular structures [1]. The localization of ALA products in subcellular structures depends on their chemical characteristics as well as on the properties of the intracellular environment [2]. Characterization of such properties is possible by means of fluorescent probes like JC-1 and carboxy SNARF-1. However, the emission spectra of these probes are overlapped with spectral pattern of typical ALA product -protoporphyrin IX (PpIX). Spectral overlap of fluorescence signals prevents to clearly separate a distribution of probes from PpIX distribution what can completely mess the applicability of these probes in characterization of cell properties. The spectrally resolved confocal laser microscopy can be used to overcome this problem. In this study, a distribution of ALA metabolic products in relation to the mitochondrial membrane potential and intracellular pH was examined. Human cell lines (KYSE-450, KYSE-70) from esophageal squamous cell carcinoma were used. Cells were incubated with 1mM solution of ALA for four hours. Two fluorescent probes, carboxy SNARF-1 and JC-1 , were used to monitor intracellular pH levels and to determine membrane potential changes, respectively. The samples were scanned by spectrally resolved laser scanning microscope. Spectral linear unmixing method was used to discriminate and separate regions of accumulation of ALA metabolic products of JC-1 and carboxy SNARF-1.

The optical properties of human cranial bones were measured in the wavelength range 800-2000 nm. The measurements were carried out using the commercially available spectrophotometer with an integrating sphere. The inverse adding-doubling method was used to determine the absorption and reduced scattering coefficients from the measurements.

We present experimental results on optical properties of cranial bone controlled by administration of propylene glycol and glycerol. Both transmittance and reflectance spectra of human and porcine cranial bone in vitro were measured. Measurements of total transmittance and diffuse reflectance have been performed using a spectrophotometer with an integrating sphere. The reflectance measurements have been carried out with an optical multichannel spectrometer with a fiber-optical probe. For estimation of absorption and reduced scattering coefficients of the bone the inverse adding-doubling method was used. The decrease of reflectance and increase of transmittance of the samples under action of the immersion agents was demonstrated. The experiments have shown that administration of the immersion liquids allows for effective control of tissue optical characteristics, that makes bone more transparent, thereby increasing the ability of light penetration through the tissue. The presented results can be used in developing functional imaging techniques, including OCT and reflectance spectroscopy.

Based on the experimental data obtained in vivo from digital analysis of color images of human irises, the mean melanin content in human eye irises has been estimated. For registration of the color images a digital camera Olympus C-5060 has been used. The images have been obtained from irises of healthy volunteers as well as from irises of patients with open-angle glaucoma. The computer program has been developed for digital analysis of the images. The result has been useful for development of novel and optimization of already existing methods of non-invasive glaucoma diagnostics.

We present experimental results on optical properties of the human skin controlled by administration ofthe 40%-glucose solution. In vivo reflectance spectra of the human skin were measured. Results of the experimental study of influence of the 40%-glucose solution on reflectance spectra of the human skin are presented. A significant decrease of reflectance of the human skin under action of the osmotic agent is demonstrated. The experiments show that administration of the glucose solution allows for effective control of tissue optical characteristics, that makes skin more transparent, thereby increasing the ability of light penetration through the tissue. Laser Doppler flowmetry has been used for study of skin blood microcirculation under the action of the glucose solution. Results of the experiments demonstrated that at the action of the glucose solution blood perfusion and blood concentration increase, however the mean blood velocity does not change. The presented results can be used in developing functional imaging techniques, including OCT and reflectance spectroscopy. A potential benefit of the optical clearing technique is the improvement of laser therapeutic techniques that rely on sufficient light penetration to a target embedded in tissue.

We report the use of light scattering to monitor the morphological changes in mammalian breast cancer cells (MCF-7) undergoing UVA irradiation induced apoptosis. The values for the scattering anisotropy parameter (g) and reduced scattering coefficient [μ_s^/ = μ_s (1-g), where μ_s is the scattering coefficient] of the control cell suspensions and cell suspensions irradiated to varying dosage of UVA (80 - 160 kJ / m²) were estimated for the wavelength range 450nm - 750 nm using integrating sphere measurements of diffuse reflectance and transmittance. These estimates were used to determine the Mie equivalent scatterer radius (r _Mie) and thus quantify the morphological alterations of the cells. The value for r _Mie was observed to decrease monotonically with increase in the UVA dose to up to a dose of 140 KJ/m² and showed an increasing behavior beyond this dose. Since it is known that while apoptosis leads to shrinkage in volume of the cells and fragmentation of nuclei, necrosis of the cells is associated with a swelling of cells, the results suggest that at doses below 1 40 kJ/m² cells undergo apoptosis and beyond this dose necrosis takes place. We also used the light scattering approach to monitor effect of He-Ne pre-irradiation on UVA induced damage in cells. Results were consistent with previous reports that suggest that He- Ne pre-irradiation can induce protection against the UVA damage to the cells.

In this work the version of indirect iterative methods for determination of optical parameters of biological tissues is presented. This method determines such optical parameters as anisotropy factor (g), absorption coefficient (μ_a), scattering coefficient (μ_s). Both these parameters determine mean free path (d_s) which use in simulation of light propagating trough biological tissue. For light propagation modeling we used weight time-resolved Monte Carlo method (MCM), time-depended diffusion approximation method (DA), and hybrid method (MCM-DA). Three these methods can use in different cases. The DA is less accurate and universal technique than MCM but it, in contrast to MCM, doesn't need large computational burden. Hybrid model has advantages of both methods. We found dependences of transmitted through biotissue pulse spreading with which optical parameters are determined. In this study we didn't take into account femtosecond pulse spreading at the expense of refractive index dispersion.

In this paper we develop the concept of a living optical phantom that uses engineered tissue as a phantom for calibration and optimization of biomedical optics instrumentation. With this method, the effects of biological processes on measured signals can be studied in a well controlled manner. To demonstrate this concept, the effects of cellular remodeling of a collagen matrix on the optical scattering properties were investigated using optical coherence tomography (OCT). Living optical phantoms of the vascular system were created by seeding smooth muscle cells in a collagen matrix. The optical scattering properties (scattering coefficient μ_s and effective anisotropy factor g_eff) were extracted from OCT images through mathematical processing. We found that the scattering coefficient of a remodeled matrix was generally higher than that of an unmodified matrix. The results indicate that OCT may provide meaningful information on how cellular remodeling of an extracellular collagen matrix changes its scattering properties. More broadly, we believe that making such optical measurements on living optical phantoms can help define the potential of biomedical optics technologies for studying biological systems.

On the basis of studying of opportunities of Reflectance Tissues Oximetry (RTO) as well as on the basis of RTO and Laser Doppler Flowmetry (LDF) comparative data, received in Moscow Regional Research and Clinical Institute "MONIKI" while patients with the peripheral blood microcirculation disorders were under examination, it was offered to unite the RTO and LDF techniques in a single diagnostic system. The new two-channel diagnostic system will contain the first LDF channel to measure the blood microcirculation parameters and the second RTO channel to register an average peripheral blood oxygenation. In the report the features of the new system design and a number of experimental data on correlation of RTO and LDF results are considered. The prospects of amalgamation of these two different techniques in a single diagnostic device are shown as well.

Reduced ability of erythrocytes of ischemic rats to change their shape in shear flow and semax effect on the deformability of erythrocytes are studied with laser diffractometry technique. It is shown that both in vivo and in vitro applied semax positively influences the impaired deformability properties of erythrocytes of ischemic rats.

Different methods of speckle-metrology, which may be used in biomedical diagnostics, are considered in this paper. Cross-correlation technique, digital speckle-photography, LASCA are compared. Advantages and disadvantages of these methods are demonstrated; the limits for minimal resolution are studied.

One of the important problems of a bleeding gastroduodenal ulcer surgery is a prognosis of the recurrent hemorrhage and appraisal of endoscopic hemostasis quality. Endoscopic Laser Doppler Flowmetry of a mucous coat of stomach and a duodenum was made on 34 patients for the purpose of investigation of features of microcirculation. Analogous researches are made on 30 patients with a peptic ulcer and on 28 practically healthy people. Analysis of LDF-grams has shown certain differences in regional microcirculations in stomach and duodenal at normal and at a pathology. Increase of regional perfusion in periulcerose zone with its pathology disbalance can serve as a criterion for activities of an alteration processes in gastroduodenal ulcer defining the risk of possible hemorrhage.

In order to increase the spatial resolve of measuring the optical properties of local tissue, the source-detector separation should be decreased. However, in this case, the diffuse approximation theory is invalid to describe the photon migration of local tissue, and could not use to attain the optical properties of tissue from the reflectance measurements. The Monte-Carlo method is used to simulate the photon migration in turbid media, but the inverse problem is very complex. Therefore, we try to give an approximation formula to describe the photon migration in this condition and attain the optical properties. In this paper, a novel optical detecting system was introduced to measure the optical properties of local tissue, which employed 1-D fiber array, and the maximum of light source-detector separation to collecting fiber can be confined in 1 mm. Based on the measurement of the different concentration intralipid, and an empiristic model was established to show the relationship between the separator-dependant reflectance and optical properties, which well agreed with Monte-Carlo simulation. Ulteriorly, the optical properties were derived from the empiristic model, the error from this approximation theory were analyzed. The results demonstrate that the empiristic theory can be used to describe the photon migration in local tissue and rapidly acquire the optical properties of local tissue.

The inhomogeneity of tissue structure because of individual differences greatly affects the sensitivity of tissue oxygenation saturation measurement by reflectance NIRS. In this study, a continue wave NIRS system was developed, which had parallel arranging one source and multi-detectors. An LED incorporated three elements of 735nm, 805nrn, 850nm peak wavelengths was used for the source. There were three detectors and a source, and the detector-detector separations were 5mm, and the source-detector separations could be changed. Ischemia tests on the forearm were performed on six male and six female subjects (22-27 years of age and 45-75 kg in weigh). 80mmHg was vein ischemia test and l20mmHg press was for whole ischemia test. Meanwhile, the fat layer thickness was also measured. According to the fat thickness of the forearm, the tests are divided to three groups, the fat group, the standard group and the thin group. The results showed that the measurements of muscle oxygenation depended widely on the source-detector separations and the individual. There are less relative errors of muscle oxygenation among one of the groups than those among all of the groups. The measurements were different due to different thickness of fat layers under the same source-detector separations. The thicker the fat layer, the more obvious the muscle oxygenation changes for different tests under the same source-detector separation. Therefore, the optimal source-detector separation must consider the fat thickness for measurements of muscle oxygenation by NIRS.

This paper compares sensitivity to blood oxygenation of different schemes of detecting light scattered from or transmitted through a slab of tissue considered in the frames of a multilayer model. Comparison is made from the viewpoint of the sensitivity to oxygen saturation of certain blood volume confined within lower layers of different average thicknesses mimicking the upper and lower plexuses of skin, dermis and hypodermis. The model also includes upper layers, mimicking stratum corneum and epidermis, consisting of prickle and basal cell layers. The following signals were simulated with Monte Carlo technique and compared: diffuse scattering indicatrice, OCT signal, spatially resolved diffuse reflectance, time-of-flight and spectrophotometry signals. The optical parameters of the layers were chosen within the ranges corresponding to experimental data published in literature and our own OCT measurements. Heyney-Greenstein function was used as a phase function for all considered layers with anisotropy factor value varying for various layers. Blood fractions of different layers were chosen according to available data for diastolic state. We considered two wavelengths of 660 and 890 nm, located at different sides of the isobestic point of 805 nm, where the absorption coefficients of oxygenated and deoxygenated hemoglobin are equal. These wavelengths are used in pulse oximetry. Our simulation results show, that the highest sensitivity to changes in oxygen content in blood is at the wavelength of 660 nm, where the difference between absorption coefficient values is significant. For this wavelength all the techniques except OCT show good sensitivity to blood oxygenation in the model tissue. For the second wavelength goniophotometry, spatially resolved diffuse reflectance, and spectorphotometry exhibit sensitivity to oxygenation, but it is lower than for 660 nm due to a smaller absorption coefficient mismatch.

Controlling of optical properties of skin is of great interest for skin diseases monitoring and phototherapy. Glycerol, as the most effective optical clearing agent, was applied in this study. Effects of osmotic properties of glycerol on optical clearing of the human skin have been investigated. Two main mechanisms of optical clearing at a creation of lattice-like pattern of localized thermal damage islets in the SC, glycerol diffusion into damaged skin and tissue dehydration, were discussed.

The problem of pencil optical beam reflectance by semi-infinite biological media is considered using microscopic hybrid approach to the radiative transfer equation (RTE). This approach is based on the reciprocity of the Green function for the radiative transfer theory and the iteration procedure of the solution to the integral equation for Green function written in reciprocal form. As a result the solution to the RTE is represented as a sum of two terms. The first one is diffusely reflected by biological media radiation with a finite-order scattering. The second term represents a convolution of the RTE Green function with an effective source function. The microscopic hybrid method enabled improving accuracy of the standard diffusion approximation in the problem of pencil optical beam reflectance by biological media. The relative error of the microscopic hybrid method with respect to Monte Carlo simulation is within 6% and included also the media with peak forwarded phase function of single scattering event. The main advantage of our method is that it does not use heuristic assumptions the well known in the literature hybrid models are based on. However, it is desirable to use a simple renormalization of standard diffusion asymptotics for practical applications, for example, to determine a blood oxygenation. It can significantly reduce processing time of experimental measurements. Such a simple renormalization of diffusion asymptotics is given in this work. The method lies in the selection of a factor for diffusion asymptotics at given interval distances from incident point along the surface. It is shown that acceptable accuracy for the measurements of relative values of reflectance by semi-infinite biological media is provided.

The numeric simulations of multielement capillary lens' x-ray optical properties are presented, and x-ray experiments with the lens are described. Using Cr K_α (0,229 nm) radiation and CCD-detector 11-time magnified images were obtained.

Analysis of fluorescence spectra of polycyclic aromatic hydrocarbons in human blood plasma and human serum albumin solution allowed one to conclude that pyrene and also anthracene are predominantly distributed in the hydrophobic micro-phase of blood plasma proteins. In the solution of human blood plasma containing pyrene the nonmonotonic dependence of both the intensity of pyrene fluorescence and the index of polarity on the concentration of sodium dodecylsulfate added was observed. This should be connected with the reconstruction of the structure of protein globule under the surfactant action and cannot be explained only by the solubilization of pyrene in sodium dodecylsulfate micelles.

Bacteria luciferases catalyze the oxidation reaction of the long-chain aliphatic aldehyde and reduced flavinmononucleotide involving molecular oxygen to a respective fatty acid emitting light quanta in the visible spectrum. Fluorescence emission of luciferases from Photobacterium leiognathi dissolved in organic solvent-water mixtures was investigated. Methanol, acetone, dimethyl sulfoxide and formamide were used as organic solvents. As the methanol and acetone concentration is increased the emission maximum peak is decrease. In contrast, with dimethyl sulfoxide and formamide addition induced a increasing of the emission maximum intensity. The values of wavelength maximum (λ_max) at the addition of this solvent can shows the spectra shifted to the red by about 12 nm. These increasing in the fluorescence intensity and in the λ_max may be due to luciferase denaturation, resulting from the more intensive contact of chromospheres of luciferase with the solvent. At all used concentrations of methanol, acetone and formamide the shape of the fluorescence spectra was not changed. These studies demonstrate that the luciferase tryptophan fluorescence is sensitive to changes of physical-chemical property of enzyme environment. A comparison of activation/inactivation and fluorescence spectra of luciferase in methanol or acetone solutions shows that the extent of inactivation is larger than the extent of fluorescence changes at the same methanol or acetone concentration.

To begin with the present paper deals with compounds called photosensitizers, namely, psoralens (Ps), 21,22-dimethyl-10,11-cyclohexylpsoralen (DCP) and 10,11-phenyl-21,22-cyclohexylpsoralen (PhChP). The absorption spectra from excited triplets states, photophysical and spectral properties occurring in molecules were investigated in this paper. The triplet-triplet absorption spectra, spectrum S_o-S_i-absorption and rate constants of Ps, DCP and PhChP have been determined using INDO method. Experimental absorption spectra of PhCIiP were obtained using CM-2203 spectrofluorimeter.

Absence of changes of plasma proteins structure is shown by method of the crystallographic structure research that formed at drying of blood plasma at introduction in it indocyanine green. A conclusion that cases of toxic influence indocyanine cannot be connected with interaction of dye with proteins is drawn. The assumption that such cases can be caused by proteins crystallization in plasma at exceeding of admissible concentration indocyanine is put forward.

Blood plasma can be considered as a special water system with self-organization possibilities. Plasma slides as the results of wedge dehydration reflect its stereochemical interaction and their study can be used in diagnostic processes. 46 patients with coronary heart disease were studied. The main group was formed of men in age ranged from 54 to 72 years old with stable angina pectoris of II and III functional class (by Canadian classification) (n=25). The group of compare was of those who was hospitalized with diagnosis of acute coronary syndrome, men in age range 40-82. Clinical examination, basic biochemical tests and functional plasma morphology characteristics were studied. A number of qualitative and quantitative differences of blood plasma morphology of patients with chronic and acute coronary disease forms was revealed.

The problem of software and hardware implementation of methods for the visualization of fundus is considered. The parameters of the visualization must meet the medical requirements for the devices of early diagnostics of various diseases including insular diabetes, abnormalities in the permeability of the carotid artery, AIDS, etc. The requirements to the resolution and the field of registration are analyzed. It is demonstrated that the size of the registered image must be at least 2 times 10³ x 2 times 10³ TV lines (4 times 10³ x 4 times 10³ pixels). A device for measuring out-of-the-axis aberrations of the optical system of the eye (OEA) is described, which is based on the Hartmann-Shack sensor (HSS). The results of the investigation of OEA under various oblique angles and sizes of the patient's pupil are presented. It is shown that the visualization with acceptable quality within the field of the order requires 90° the size of the pupil at most 3 mm and the angular size of the registered fragment ofthe image at most 15° near the optical axis of the eye and 8° in the peripheral areas.

Ischemic heart disease is one of the leading reasons of invalidisation and death rate of able-bodied citizens in the world. There are many various surgical and medicamentous methods of its treatment for today, however all these methods have restrictions in application. Our work was directed at initiation possibility clarification of ischemic myocardium revascularization by means of making necrosis with photodynamic therapy. The investigation was carried out in rats with the ischemia artificial made by means of left coronary artery ligation. Level of Photosense photosensitizer accumulation in ischemic and normal rat myocardium zones was defined. Myocardial photodynamic revascularization procedure of ischemic rat myocardium was carried out. Morphological analysis of the myocardium preparations showed the presence of active revascularization of ischemic myocardium after photodynamic therapy. The method of ischemia level estimation based on spectral optical definition of blood oxygen saturation was developed.

Erythrocytes system study can provide a framework for detailed exploration of blood cell-cell and cell-vessel wall interactions, one of the key patterns in blood and vascular pathophysiology. Our objective was to explore erythrocytes system in patients with stable angina pectoris II f.c. (Canadian classification). The participants (N = 56, age 40 - 55 years) without obesity, glucose tolerance violations, lipid lowering drugs treating, heart failure of II and more functional classes (NYHA), coronary episode at least 6 months before study were involved in the study. Blood samples were incubated with glucose solutions of increasing concentrations (from 2.5% to 20% with 2.5% step) during 60 mm (36° C). In prepared blood smears erythrocyte's sizes were studied. Plasma total cholesterol, triglyceride and glucose levels were also measured. Received data were approximated by polynomials of high degree, with after going first and second derivations. Erythrocytes system "behavior" was studied by means of phase pattern constructing. By lipids levels all the patient were divided into five groups: 1) patients with normal lipids levels, 2) patients with borderline total cholesterol level, 3) patients with isolated hypercholesterolemia, 4) patients with isolated hypertriglyceridemia and 5) patients with combined hyperlipidemia. Erythrocytes size lowering process was of set of "stages", which characteristics differ significantly (p > 0.05) in all five groups. Their rate and acceleration characteristics allow us to detect type of lipid profile in patients. Erythrocyte system disturbing by glucose concentration increase show to be most resistant in group of patients with isolated hypercholesterolemia.

It is well known that weak electromagnetic fields of extremely high frequencies cause significant modification of the functional status of biological objects of different levels of organization. The aim of the work was to study the combinatory effect of metronidazole - the drug form of 1-(2'hydroxiethil)-2-methil-5-nitroimidazole - and electromagnetic radiation of extremely high frequencies (52...75 GHz) on the hemolytic stability of erythrocytes and hemotaxis activity of Infusoria Paramecium caudatum.

This study is focused on the determination of refractive index of hemoglobin solution at different glucose concentrations using Abbe refractometer. It is shown that the changes of refractive index caused by glycation of hemoglobin may be observed using refractive index measurements. The refractive index measurements can be potentially applied for the evaluation of glycated hemoglobin amount.

We create photonic crystal optical fiber having the photonic band gap in visible range of spectrum. This fiber consists of concentric layers of air holes of variable diameter with hollow cores. We carried out researches of their spectra of transmission. We display influence of geometrical parameters of structure on size of the photonic band gap and its arrangement in a visible range of wavelength. It is established, that variations of geometrical structure of fiber allow operating size of the photonic band gap that gives unlimited opportunities guides of light.

Influence of infrared low-level laser irradiation (LLLI) on induction of synthesis of some cytokines such as interleykin-1 (Il-1), tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), interleykin-8 (Il-8) and interleykin-4 (Il-4) by the neutrophils and macrophages in time of bacterial cells phagocytosis that was searched. As the object of analysis we used peritoneal macrophages from white mice and neutrophils from peripheral blood of healthy donors. We used the laser diod with spectrum maximum of 850 nm with doses 300, 900 and 1500 mJ (exposition -60, 180 and 300 s respectively; capacity - 5 mW). We carried out the Enzyme-Linked Immunospot Assay (ELISA) to determine cytokine content during phagocytosis after 3 h and 6 h. We found dynamics in production of the cytokines, which was different for the neutrophils and macrophages. We showed that the infrared LLLI has significant stimulating activity on the proinflammatory cytokines production by neutrophils and macrophages. Moreover we revealed dynamics changing in the Il-8 and Il-4 production.

It was revealed that irradiation of different pathogenic microorganisms by low-coherent blue light (405 nm) decreases population's quantity relatively to control for certain. Changing of biochemical activity is an important characteristic to show effect of physical action. In the present study, we have investigated the effect of low-coherent light at emission spectrum maximum near to 405 nm on bacterial metabolic activity.

Influence of low-coherent speckles on the colonies grows is investigated. It has been demonstrated that effects of light on the inhibition of cells (Francisella Tularensis) are caused by speckle dynamics. The regimes of illumination of cell suspension with purpose of devitalization of hazard bacteria, caused very dangerous infections, such as tularemia, are found. Mathematical model of interaction of low-coherent laser radiation with bacteria suspension has been proposed. Computer simulations of the processes of laser-cells interaction have been carried out. Role of coherence of light in the processes of laser-cell interaction is analyzed.

Antimicrobial photodynamic therapy strives for creation new photosensitizes providing effective formation of singlet oxygen at irradiation in various spectrum areas. Construction of a new generation of photosensitizes on the basis of nanoparticles, such as TiO₂, Ag, Au, SiO₂ have great potential. We have showed that different nanoparticles used as photosensitizes lead to different effects during photodynamic action. Interaction of broadband light and Ag-SiO₂ nanoparticle film decreased bacterial CFU. Irradiation of the test-cultures, photosensitized by TiO₂, stimulates microbial cell division.

We develop new graduate educational highly interdisciplinary program that will be useful for addressing problems in worldwide biotechnologies and related biomedical industries. This Master program called Management in Biophotonics and Biotechnologies provides students with the necessary training, education and problem-solving skills to produce managers who are better equipped to handle the challenges of modern business in modern biotechnologies. Administered jointly by Cranfield University (UK) and Saratov State University (Russia) graduates possess a blend of engineering, biotechnologies, business and interpersonal skills necessary for success in industry. The Master courses combine a regular year program in biophotonics & biotechnologies disciplines with the core requirements of a Master degree. A major advantage of the program is that it will provide skills not currently available to graduates in any other program, and it will give the graduates an extra competitive edge for getting a job then.