In laser doppler blood flowmetry (LDBFM), the flow is derived from the first moment of the power spectrum of the photocurrent. This quantity depends on both the spectral width and the modulation depth of the signal. This modulation depth depends on the amount of speckles on the detector surface. The speckle size is determined by the wavelength, the local wave form and the angular distribution of the incoming light.In theoretical treatments of laser speckle, the distance between the light source and the illuminated screen or detector is often taken much larger than the light source dimensions. In direct contact LDBFM where the detector is placed directly on or close to the skin, such assumptions do not hold, making analytic solutions of the problem impossible. The issue is studied by means of a model experiment. A scattering medium is illuminated by tow beams originating from the same laser, one of which is shifted in frequency. With a detector close to the surface of the medium, beats are observed at the difference frequencies. From the modulation depth of these beats, it is possible to derive the speckle size. The main purpose of this paper is to present this conceptually simple experiment. The first experimental results are shown. Although still of limited quality, they show the potential of the method to study the effects of scattering anisotropy, mean free path length, the detector size and its distance to the tissue.

In patients with basal cell carcinoma (BCC) skin microcirculation was measured by the laser Doppler flowmeter (LDF). Spectral analysis of LDF-signal based on fast Fourier transform enables us to evaluate an amplitude and predominated frequency of flux motions in the next diapasons: (alpha) -rhythm, (beta) -rhythm, (gamma) -rhythm, high-frequency rhythm and cardiorhythm. For photodynamic therapy (PDT) hematoporphyrin derivative was administrated intravenously in dosage 1-2 mg/Kg. In 24 hours after that all patients were exposed to the red laser irradiation in dose 200-300 J/cm². The microcirculation level in BCC was higher than in the plots of healthy skin. Exactly after PDT the marked decrease of microcirculation up to 4.7 +/- 0.63 a.u. associated with photoinduced stasis took place in the tumor. Spectral analysis of flux motions demonstrated a significant inhibition of (alpha) -, (beta) -rhythms and amplification of HF- and CF-rhythm In 1 week after PDT we observed a gradual restoration of microcirculation in the zone of PDT skin injury that followed a wound healing process.

In this paper new experimental results on monitoring of alterations of blood flow parameters in growing fish embryos under laser and non-laser light irradiation are discussed. The measurements were performed by means of laser Doppler technique with high temporal and spatial resolution. Two parameters of blood flows were mostly measured: average velocity and frequency of velocity pulsations. These parameters were shown to be an adequate characteristics of nonstationary blood flows in fishes. The problem of noninvasivity of such experiments is discussed. For this purpose absorption spectra of fish embryos were measured. The quantitative response of blood flows to irradiation at different light wavelengths was recorded. Different species of fishes were used to compare the responses. Different effects were recorded which depend upon the doze and the wavelength of irradiation, and upon the stage of the embryo development at which the irradiation had taken place. Among those effects long-term and short-term reactions can be distinguished.

We discuss the use of the QELS method for screening of population groups for verified pathologies. For mathematical analysis of experimental data the regularization procedure have been used. This allows us to determine the histograms of particle size distribution of blood plasma samples. For the interpretation of the histogram data the special program of the mathematical processing - 'semiotic classifier' - have been created. The main idea of the 'semiotic classifier' is based on the fact, that formation of the pathological trace in human organism depends not only on concrete disease nature but also on the interaction between the organism sanogenetic mechanisms. We separate five pathological symptomatic complexes of organism status: allergic diseases, intoxications, organism catabolic shifts, auto-immune diseases and degenerative-dystrophy processes. The use of this 'semiotic classifier' in the system of monitoring investigations allows to solve the next problems: (1) to separate the persons with the expressed initial level of pathological processes to the risk groups for the special clinical investigations, (2) to set up the predisposition of the concrete individual towards definite pathologies at the preclinical stage, (3) under the conditions of expressed clinical pathology to study the dynamics of pathology processes.

We describe a noninvasive technique and instrumentation for measuring the oxygen saturation of blood in retinal arteries and veins. A white-light image of a subject's retina is presented to an operator. The operator targets a particular retinal artery or vein and initiates the measurement procedure. The measurements are made by scanning low-power lasers into the eye and across the retinal vessel. The light reflected and scattered back out of the eye is collected and measured. The oxygen saturation of blood within the vessel is spectroscopically determined by analyzing the vessel absorption profiles at two or more wavelengths. A complete saturation measurement can be made in less than one second, allowing real-time measurement during physiological changes. The sensitivity of this measurement technique to changes in retinal saturation has been demonstrated through a series of pilot studies in anesthetized swine. We present data indicating that retinal venous oxygen saturation decreases predictably during ongoing blood loss, indicating a potential application of an eye oximeter to nonivasively monitor blood loss. Current invasive techniques for monitoring bleeding, such as fiber optic pulmonary catheters, are not suited for use early in trauma situations. A portable eye oximeter may therefore provide a new technique for reducing mortality in the emergency department setting.

Native fluorescence spectroscopy of biomolecules has emerged as an intrinsic parameter in the characterization of the physiological state and the discrimination of pathological from normal conditions of cells and tissues. The key fluorescing biomolecules inc ells and tissues ar tryptophan, tyrosine, phenylalanine, collagen, elastin, NADH, flavin and porphyrin. Extensive studies were made on tissues of various origin to discriminate the malignancy from normal. The differences in the fluorescence emission spectra have been shown to separate benign and malignant tissues. In the present work, a pilot study was carried out on the characterization of blood plasma of both normal and cancerous subjects. The blood plasma was separated by centrifuging the blood and it was diluted in PBS by adjusting the O.D. to 0.5 at 280 nm. This diluted sample as excited in the UV region between 250-340 nm. Among the various excitation wavelengths, emission spectrum at 300 nm excitation has considerable difference between blood plasma of normal subjects and cancer patients. To quantify these differences and to verify if there is any diagnostic potential exists, the ratio of fluorescence intensities at 340 and 440 nm was calculated. It is found that the ratio value of normal blood plasma is less than 11 and for tumor, it is greater than 11. Besides, it is found that the ratio value of blood plasma from patients with cancer varies from 11 to 28, depending upon the stage of malignancy.

In recent years investigation and understanding of the brain activity is receiving much attention. Such investigations are generally confined to few select premier research institutions where expensive and sophisticated facilities like EEG, PET, FMRI, etc. are available. Of late optical sensors are receiving much attention for biomedical applications because they are relatively simple in construction, easy to use and comparatively inexpensive. Among the biomedical optical sensors, photophlethysmographic (PPG) measuring systems have a unique position. They function as transcutaneous registration of blood volume changes in the near skin blood vessels. By recording the signals from the supply to the left and right lobes of the brain in the cerebral cortex. The oxygen content in the arterary blood flow to the brain will naturally have an important role to play in the activity of the brain. It is suggested that by positioning sensitive temperature sensors in the nostrils of a subject, one could monitor his breathing activity. By recording the outputs rom these temperature sensor for several hours, it has been noticed that the breathing activity of a subject will change from one nostril to another periodically. Besides, it has also been observed that any sudden fluctuations in the breathing pattern is accompanied by changes in the blood flow to the brain as monitored by PPG optical sensors mounted on the temples of a subject. An attempt is made to understand such events.

Parameters of chemiluminescence (CL) from nondiluted human blood were studied. Kinetics and intensity of CL depended upon donor's state of health, time after blood extravasion, conditions of its storage, conditions of its counting. Peculiar dependence of Luminol-enhanced CL on sample volume changes during respiratory burst (RB) was revealed. When 0.5 ml aliquots were consecutively taken from blood and transferred into another vial of the same configuration, each subtraction of blood was followed by an acceleration of CL intensity growth. Summation of portions of blood in the second vial resulted in deceleration of CL intensity increase from it. At equal volumes of blood CL intensity from the first sample was manifold higher than from the second one and this difference was increasing on with further transfers. When blood was transferred back to the first sample, CL intensity from the 'donor' sample began to increase at a faster rate, while CL intensity from the 'recipient' sample stabilized. Such behavior was characteristic of nondiluted healthy donors' blood. Diluted blood or blood of sick people demonstrated different behavior. It is suggested that CL parameters of nondiluted blood may be informative of integrative properties of blood tissue.

The time course of hemolysis by a number of amphiphiles differing in their molecular features was studied in isotonic solution under varying temperature and concentrations of amphiphile and erythrocytes. Some of detergents induced hemolysis with two-stage kinetics, the property not correlating with either the detergent polar group charge or critical micelle concentration. The first stage appears at relatively high amphiphile concentrations, starts without a notable lag-phase and stops within less than 1 min at an intermediate extent of lysis; the second stage has a distinct lag-phase followed by the exponential lysis phase always reaching complete hemolysis.The parameters of the two stages differently depend on temperature and concentration of sucrose. A general two- stage scheme of amphiphile-membrane interaction is proposed. During the former stage, amphiphile molecules, bound initially on the outer surface of the cell membrane, equilibrate across the membrane. At a sufficiently high amphiphile binding per cell, the first stage hemolysis occurs with the rate decreasing sharply in the course of transmembrane equilibration. After reaching the equilibrium distribution of detergent across membrane, hemolysis follows the second stage mechanism.

Bio-speckle flowmetry is useful for measuring the blood flow velocity in human retinal vessels, but it often suffers rom eye-movement artifacts and other external moving factors. To solve this problem, we studied in this paper here a flexible correlation-analyzing system which is able to extract effectively the blood flow information from erroneous data influenced by a displacement of the measuring point. On the basis of the photon correlation technique, the system directly stores the sequential counts of photo-electron pulses into the memory. After a measurement, the stored data are read out and used to calculate the first-order statistics of time-integrated speckle fluctuations and the autocorrelation function with an arbitrary delay-time unit for an arbitrary period. These results are used to specify the displacement of the measuring point due to eye movements and to eliminate erroneous data. The usefulness of this technique was verified for a glass capillary model and human retinal vessels.

A study on the multiply-backscattered light with low scattering orders form a strongly absorbent medium where the light can hardly propagate is of benefit to the metrology in biophysics, chemistry, and others. This report is concerned with the investigations on a spatial intensity distribution and a temporal autocorrelation function of the time-varying light backscattered coherently from aggregated particles in uniformly random and fractal media. The investigations were conducted by means of Monte Carlo simulation based on the Rayleigh-Debye scattering theory. In the spatial intensity distribution, the peak intensity of an enhancement cone decreases for the co-polarized intensity but increases for the cross-polarized intensity with an increase of the absorption while its width for both cases spreads. A fractal dimension of the medium affects a decreasing rate of the intensity cone, independently of the absorption. In the temporal correlation, the absorption influences a decay of the autocorrelation function in the short delayed time in such a way that the relaxation time increases consistently with the absorption. A decreasing feature of the autocorrelation function the long delayed time depend on the fractal dimension rather than the absorption but its effect is not so remarkable. In the report, we reveal the spatial and temporal features of coherently-backscattered intensity variations with relation to the occurrence rates of the pathlength and the scattering order in the light propagating from the incidence to the exit.

Dynamic light scattering studies show that concentration and pH play important roles in determining pig gastric mucin's (PGM) ability to aggregate and gel. At low concentrations, PGM macromolecules exist in solution predominantly in the form of monomers. At high concentrations, PGM macromolecules aggregate to form supra-macromolecular clusters. When the pH of the high concentration PGM solution is changed from 7.0 to 2.0, the system undergoes a sol-gel transition: from a solution of polydisperse aggregates to a gel. This pH and concentration dependent sol-gel transition of PGM solution may provide a mechanism for the mammalian stomach to protect itself against being digested by the gastric juice.

The instrument for the quasielastic light scattering (QELS), LCS-03 utilizes heterodyne optical scheme which permits a high resolution determination of particle size distribution. The vibration related problems, which are common for the heterodyne techniques, have been overcome by using a single glass block incorporating all the optical elements. The real-time correlation analysis of the photocurrent fluctuations is performed by a PC-embedded analog-to-digit converter card with digital signal processor (DSP) using an original algorithm. Both the technical specifications of the instrument and the software for the size distribution analysis are presented. The heterodyne technique consistently outperforms the homodyne one when the accurate characterization of the particle size distributions in heterogeneous systems is required. Diagnostic analysis of size distribution of particles in blood serum/plasma, liquor and saliva is such an application. This kind of diagnostics usually requires a simultaneous analysis of huge number of QELS data. The original statistical algorithm with graphic user interface is described. We discuss the technical specifications of instrumentation as well as methodical problems of biological fluids QELS diagnostics.

The influence of microwaves on biological materials, water solutions and pure water is the subject of many experiments and theoretical investigations. Some aspects of this problem include the so-called 'non-thermal' effects and arising of metastable states in liquid water under the action of microwaves. We carried out experimental investigation of the thermal effects of microwave interaction with water solution. These experiments consist of the spectrophotometric investigation of the reaction of non- organic water solution on the influence of 25 GHz microwaves with the thermal intensity. We investigate the optical density of the 12 percent MgSO₄ water solution, heated by microwaves to the different temperatures: 40 degrees C, 30 degrees C, and 25 degrees C. Water irradiated by low intensity continuous microwave radiation also exhibits an increase in optical density in near UV region, caused by the excitation of the electronic configuration of water molecule. The results obtained and therefore, indicative of changes in the water molecule itself under the action of microwave irradiation. The optical density spectra show these changes to be long-living.

The basic concepts of biofluids crystallooptic diagnostics (COD) are described: crystallization texture reflects the complex of biofluid physical and chemical properties. The informational potential of COD is discussed. Combining of COD with the modern computer technologies transfer this technique into the category of intellectual prompts which supply the standardization of analysis and understanding of images and are able to complete the modern tomography techniques in future.

Ultraviolet resonance Raman spectroscopy (UVRRS) has the potential to become a sensitive, specific, versatile bioanalytical and biophysical technique for routine investigations of proteins, DNA, and their monomeric components, as well as a variety smaller, physiologically important aromatic molecules. The transition of UVRRS from a complex, specialized spectroscopic method to a common laboratory assay depends upon several developments, including a robust sample introduction method permitting routine, in situ analysis in standard laboratory environments. To this end, we recently reported the first fiber-optic probes suitable for deep-UV pulsed laser UVRRS. In this paper, we extend this work by demonstrating the applicability of such probes to studies of biochemical relevance, including investigations of the resonance enhancement of phosphotyrosine, thermal denaturation of RNase T1, and specific and non-specific protein binding. The advantages and disadvantages of the probes are discussed with reference to sample conditions and probe design considerations.

Micro-interferometry, a novel technique developed by the authors, employs a linearly polarized laser, a fused silica capillary tube housing for the sample and a charged coupled device as a detector. A back scattered interference pattern, observed as a high contrast fringes, is produced when the laser is directed onto the capillary containing the sample. The positional change of the fringe pattern is a function of the refractive index of the media in the capillary. In the present work, the RNA enzyme RNase T1 is heated in the sample cell over a temperature range of 30 degrees C to 60 degrees C. Over this temperature range the molecule unfolds form the quaternary to the tertiary structure. This structure change is manifested as a refractive index change and is observed by monitoring the fringe position while ramping the cell temperature in a controlled fashion. From the refractive index response over the temperature range, the Gibbs free energy associated with unfolding is calculated. The authors show milli-degree temperature stability with a 0.1 micro-liter probe volume, thus demonstrating the application of this device in micro- calorimetric investigations.

An antibody-based magnetic plate chemifluorescent immunoassay (MPFIA) for effective and rapid detection of bacteria and toxoid from biological fluids was developed. Streptavidin (SA)- magnetic particles and biotinylated antibody as a solid phase immunomagnetic carrier was used for antigen capture. An alkaline phosphatase-antibody conjugate as a secondary capture antibody to the antigen forms a sandwich with the primary antibody. The fluorgenic substrate, AttoPhos reacts with alkaline phosphatase that emits chemifluorescent intensities are proportional to captured antigens. Antigen separation and concentration from biological fluids using immunomagnetic carrier are the key step to reduce media interference for sensitive detection. Results of these efforts may actually enhance the immunoassay possibilities by concentration of specific antigen and reduction of background noise. Magnetic separation and chemifluorescent detection have been achieved by a multiple-well formatted magnetic plate separator and a fluorescent plate detector, respectively. Experiments were performed for virulent Escherichia coli cells, Staphylococcal enterotoxin B toxoid and Bacillus subtilus spore detection in biological fluids. In general, the fluorescent detection can be achieved at the same sensitivities as enzyme-linked immunoassay and the ECL detection is more sensitive than fluorescent assay. However, the unique features of MPFIA and MPECL are that the biological samples can be rapidly processed and detected on the same multiple sample formatted plate within one hour assay time.

Many techniques have been developed to investigate the chemistry associated with brain activity. These techniques generally fall into two categories: fast techniques with species restricted sensitivity and slow techniques with generally unrestricted species sensitivity. Therefore, a need exists for a fast non-invasive technique sensitive to a wide array of biologically relevant compounds in order to measure chemical brain events in real time. The work presented here describes the progress made toward the development of a novel neurotransmitter probe. A fiber-optic linked Raman and tunable ultraviolet resonance Raman system was assembled with custom designed optical fiber probes. Probes of several different geometries were constructed and their working curves obtained in aqueous mixtures of methyl orange and potassium nitrate to determine the best probe configuration given particular sample characteristics. Using this system, the ultraviolet resonance Raman spectra of some neurotransmitters were measured with a fiber-optic probe and are reported here for the first time. The probe has also been used to measure neurotransmitter secretions obtained from depolarized rat pheochromocytoma cells.

Near-IR spectroscopy has been used in combination with multivariate calibration techniques such as partial-lest squares regression (PLSR) to quantify glucose concentration in various media. However, for reasonable prediction capability in measuring glucose many calibration samples are needed. n addition, spectroscopic data often contain over 1000 data points, presenting a very large data matrix for calibration. It is desirable to reduce the available data to contain only the information necessary for accurate prediction of chemical concentration before PLSR is applied. This will eliminate noisy variable sand consequently the data can be processed more quickly and efficiently. A variable selection method that reduces prediction bias in single factor partial least square regression models was developed and applied to near-IR absorbance spectra of glucose in two different media: pH buffer and cell culture medium. Comparisons between calibration and prediction capability for full spectra and reduced sets were completed, resulting in statistically equivalent mean squared errors. The number of response variables needed to fit the calibration data and accurately predict concentrations form new spectra was reduced in each case. The algorithm correctly chose the glucose peak areas as the informative variables and computation time was decreased by an order of magnitude.

It was found that distilled water possesses weak luminescence in the near UV and visible regions of the spectrum. The excitation spectrum is complex and has two main maxima, at 270 and 310 nm. The corresponding emission spectra apart from a narrow lines due to Raman scattering are represented by wide lines at 360 and 410 nm and are determined by the inherent properties of water. The intensity of luminescence depends on the time of holding of a sample in a closed vessel and the addition of a small amount of both luminescent and non-luminescent dipeptides. The observed phenomena can not be reduced to the luminescence of admixtures in water, but is a result of unique properties of water, its structure and polymorphism. The effect of water 'hardening' occurring upon rapid cooling of a hot sample to a room temperature, in contrast to slow cooling, is discovered. It is attributed to the formation of a new steady state of an aqueous structure, indicating by very intensive luminescence band at 5450 nm. Relative intensities of the described bands of emission are greatly sensitive to weak fields of electromagnetic nature. The phenomena observed lead to the conclusion that water and aqueous solutions should be regarded as a continuous polymorphous containing defects structures which are in general non-equilibrium self-organizing systems.

Samples of erythrocyte suspensions of normal and pathological blood were studied by light back-scattering technique. This method allows to register the kinetics of the formation of erythrocyte aggregates in stasis and the disruption of large and small aggregates in shear flow of aggregated blood in a Couette chamber. The output signal proportional to backscattered light intensity was processed by different algorithms to quantitatively determine the aggregational properties of erythrocytes. We suggest that the registered time dependence of the signal representing the aggregation kinetics can be approximated as a sum of exponentials with parameters T₁ and T₂ that reflect the characteristic times of linear and clump or network aggregates formation respectively. On the other hand the hyperbolic approximation of the aggregation kinetics enables to determine the characteristic constant of the whole aggregation process. We show that the parameters obtained with both exponential and hyperbolic data processing algorithms are sensitive to the concentration of erythrocytes in blood sample, mode of scattered light detection and other factors. The advantages of each algorithm and latest results of their comparative analysis will be discussed in this paper.

Properly measured experimental Mueller matrix contains complete information on depolarization, anisotropy properties of studied object and on value of isotropic change of probated radiation intensity by studied object as well. They know that Jones matrix contains complete information on value of isotropic change of probated radiation intensity and on anisotropy properties of studied object. Thus, in the case of absent of depolarization and measurement errors reducing to existence of, so called, overpolarization there exist a one-to-one correspondence between Mueller and Jones matrix. Mueller matrix will then be called a Mueller-Jones matrix. The possibility of extraction of Mueller-Jones part out of any experimental Mueller matrix is extremely important because of following. First, it allows us to obtain everything about depolarization properties of studied object directly. Depolarization is very informative 'object' and now, in the majority, one knows little about its nature and methods of its complete description. Second, one gets the possibility to operate with correspondent Jones matrix to analyze of which there exist the powerful methods such as solving of the spectral problem and application of the decomposition theorem formerly proved by the present authors. The distinctive feature of the method proposed here is that it allows us in the best way to take into consideration the important fact that far from all elements of initial Mueller matrix contains information on depolarization.

Blood parameters and its intervals of changes and the methods of their registration were considered. A coherent optical method of analysis of blood cell parameters was considered. Average statistical parameters and form features of the cells and its nuclei can be determined from spatial- frequency spectrum analysis. The relationship between the blood parameters and coherent optical parameters has been determined. The model of the analyzer and the experimental results are described.

The unique analytic potential of biofluids crystallooptic diagnostics (COD) is determined by visualization of aggregation properties and molecular biofluid organization, that reflect an important information about functional state of separate systems as well as about the physiological status of the whole organism. Extraction, visualization and processing of the diagnostic information are supplied by the smart-technology. COD techniques experience in studies of bile, urine, liquor, tear, saliva, blood and other biological fluids is generalized: crystallooptic diagnosticums are the pool of analytical system 'Mesotest'. Combining of biofluids COD with the modern computer technologies transfer such methods into the category of intellectual prompts.

An instrument has been designed and implemented capable of mapping oxygen distribution in skin tissue over an area of several square centimeters with a spatial resolution of better than 1 mm and with a resolution in oxygen partial pressure of better than 5 torr. The measurement scheme is optical and is based on luminescence lifetime. It is non- invasive and avoids any patient contact with electrical parts. The instrument should be a valuable supplement to other clinical methods for monitoring microcirculation and peripheral oxygen supply.

We have developed fiber-optic probes that facilitate rapid, simultaneous determination of multiple analytes, in situ, over a broad range of concentrations. Theoretical and empirical methods were used to design and characterize prototype probes that comprise a single small-diameter excitation fiber and multiple larger diameter collection fibers for the optical collection of side- and back- scattered or emitted light, depending on the sample characteristics. Prototype were developed for use with pulsed ultra-violet resonance Raman spectroscopy, however, probes of this type are also suitable for use with other spectroscopic techniques such as fluorescence. Materials specifications, modelling methods, fabrication methods, and performance characteristics are described. Probes of our design are at present capable of measuring the aromatic amino acids in the 10 (mu) M range and nM detection limits can be expected. We have also obtained UV Raman and resonance Raman spectra from proteins, DNA, amino acids, steroids, neurotransmitters, and alcohols with these probes.

Resonance Raman spectroscopy, performed via an optical fiber probe, can be used in aqueous samples to detect a wide variety of chemical species in situ. It offers a potentially rapid, on-site alternative to the high performance chromatography/mass spectrometry methods currently considered definitive for the detection of sex steroids in human urine. As a first step in the development of a resonance Raman instrument for the rapid detection of sex steroids in biological samples, it had to be shown that these substances, their analogs, and the major components of human urine can be differentiated on the basis of their resonance Raman spectra. A fiber-optic linked Raman and tunable ultraviolet resonance Raman system was assembled with custom designed optical fiber probes. The ultraviolet absorption spectra of some sex steroids, analogs, and components of human urine were measured in order to determine feasible excitation light frequencies. We present here for the first time the UV resonance Raman spectra of these substances obtained via our novel fiber probes. These results indicate that some of the steroids tested can be differentiated from each other and from the major components of human urine on the basis of their resonance Raman spectra.

At the Department of Biomedical Engineering of Drexel University, the study of the reactivity of human white blood cells (leukocytes) had been undertaken originally for biocompatibility measurements. Acute (jolymorphonuclear) and chronic (mononuclear) inflammatory white blood cells (leukocytes) have been studied for their response to chemoattractant and chemorepellant implantable materials. Further experiments have lead to the enhancement and retardation of this cytotaxis via electromagnetic fields. These techniques are now being adapted to study the photonic effects of leukocytes.

Recently more attention has been given to the role of the lysosomal enzymes in the development of various pathological conditions. There were indications on the participation of lysosomal ferments in the development of various eye diseases such as glaucoma. Studies on the (beta) - galactosidase and cathepsins B and D activity in the blood's serum of patients with primary open-angle glaucoma were conducted. The activity of enzymes was determined by the spectrophotometry method. Cardiovascular and chronic lungs diseases accompanied ones in the main group. Patients included in the control group were of the same age and with the similar somatic patholegym. Statistically authentic galactosidase activity in the serum of patients with glaucoma as compared with control group was statistically unauthentic. Cathepsin B activity in the serum of patients with glaucoma was authentically increased at all stages of disease. In the contrary, cathepsin D activity increased at stages 2 and 3 in comparison with the control. On the basis of these studies we come to conclusion that primary glaucoma is accompanied by the increase of activity of cathepsins B and D in the serum.

In order to understand the mechanisms of human sclera optical enlightening due to administration of osmolytes in vitro investigations of time-dependent transmittance spectra of bovine sclera were undertaken.

The known models do not provide satisfactory description of the erythrocyte sedimentation kinetics. Experimental data have shown that erythrocyte sedimentation rate (ESR) depends on (i) hematocrit (H); (II) aggregation degree; (iii) height of the liquid column (h_o); (iv) the state of the capillary inner surface and (v) diameter of the capillary (D). The latter dependence is very strong at D approximately 1 mm. Such an effect is due to a notable influence of rubbing forces on the sedimentation process at small D. Significant scatter of the ESR data for a variety of healthy donors may be due to non-standard laboratory capillaries. In this work, a mathematical model has been developed for the case of D > 4 mm, the contribution of friction being negligible. According to experimental data, the dependence of the initial ESR value (v_o) on H is hyperbolic at H yields 1 and linear at H yields o: v_o equals 2gR²(Delta) (rho) (1 - H)/9(eta) (H), where g equals 9.8 m/s², R is the average erythrocyte radius, (Delta) (rho) is the difference between specific densities of erythrocytes and the medium, (eta) (H) equals (eta) _o (1 + (alpha) H²) is the viscosity of erythrocyte suspension at H < 0.4, (eta) _o is the medium viscosity. A kinetic equation has been derived describing the time dependence of the height of erythrocytes column, h: h-h₀ + Hh₀ (1 + (alpha) ) 1n (h-Hh₀) / h₀ (1-H)) equals - At, A equals 2gR² (Delta) (rho) /9(eta) ₀. The equation is in a good agreement with experimental data.

Results of Monte-Carlo simulations of Doppler shift are presented for model for random medium that containing moving particles. The single-layered and two-layered configurations of the medium are considered. Doppler shift of the frequency of incident laser light is investigated as a function of such parameters as absorption coefficient, scattering coefficient, and thickness of the medium. Possibility of application of speckle interferometry for diagnostics in dentistry have been analyzed. Problem of standardization of the measurement procedure have been studied. Algorithm of processing of Doppler signal in the diagnostics of blood microcirculation in mucous membrane have been suggested.

The use of polarimetry in the investigation of chiral molecules has been researched for over a century. However, it has not been until recently that the sensitivity and accuracy of this technology has improved enough to be applied to the quantification of the low optically metabolite concentrations seen within the body. The long term goal of this research is the development of a polarimetric detection system with presence of other confounders. In this study, a robust polarimeter utilizing digital closed-loop control was designed and constructed that can effectively measure millidegree rotations of plane- polarized light within biological media. In vitro experiments were conducted using a 1 cm path length sample cell in both glucose doped cell culture and bovine aqueous humor media with analyte concentrations on the order of those seen in the body. A high degree of linearity between the measured signal and glucose concentration is seen during calibration of both the cell culture and aqueous humor experiments with correlation coefficients of 0.9995 and 0.9912, respectively. In addition, validation of the obtained calibration models yielded standard errors of prediction of 8.469 and 20.25 mg/dl for each media, respectively. Overall, we feel the conducted experiments are a logical step to furthering the development of using polarimetry in the detection of optically active metabolites, and our results indicate that accurate detection of glucose in the presence of additional confounders can be accomplished in both cell culture and aqueous humor media.

We apply backscattering nephelometry technique to register the alterations of the scattering signal from a whole blood sample due to appearance or disappearance of different types of erythrocyte aggregates in stasis and under controlled shear stress. The measured parameters are: the characteristic times of linear and 3D aggregates formation, and the strength of aggregates of different types. These parameters depend on the sample temperature in the range of 2 divided by 50 degrees C. Temporal parameters of the aggregation process strongly increase at temperature 45 degrees C. For samples of normal blood the aggregates strength parameters do not significantly depend on the sample temperature, whereas for blood samples from patients suffering Sjogren syndrome we observe high increase of the strength of 3D and linear aggregates and decrease of time of linear aggregates formation at low temperature of the sample. This combination of parameters is opposite to that observed in the samples of pathological blood at room temperature. Possible reasons of this behavior of aggregation state of blood and explanation of the observed effects will be discussed.

Several parameters of the cardiovascular system such as heart rate, arterial blood pressure and blood flow fluctuate spontaneously due to the autonomic nervous system activity. In the current study, the low frequency fluctuations of the tissue blood volume and the blood volume pulse in the fingertips of healthy subjects were investigated using transmission photoplethysmography (PPG). The baseline of the PPG signal (BL) is inversely related to tissue blood volume so that the parameter BV, defined by: BV equals Const.-BL is directly related to the blood volume. The amplitude (AM) is directly related to the systolic blood volume increase. For most of the examinations BV and AM show positive correlation, which is expected since BV depends on the tissue blood volume and AM depends on the compliance of the blood vessels, both of which decrease during vasoconstriction, which is caused by higher activity of the sympathetic nervous system. The analysis of the PPG signal provides, therefore, a potential tool for study in the mechanism of the regulation of the microcirculation by the sympathetic nerves.

The orthogonal axes of illumination, flow, and detection in conventional sorting flow cytometers can limit accuracy or throughput when making fluorescence measurements on a spherical cells. A new radially symmetric optical configuration has been designed to overcome these problems. Both illumination and fluorescence collection are performed by a single optical element which encircles the sample stream flow axis. Unlike existing epi-illumination flow cytometer designs, these optics are compatible with electrostatic sorting. The resolution of this system is currently being evaluated for DNA chromosome content measurement with an ultimate goal of separation of X- and Y- chromosome-bearing mammalian spermatozoa. We describe the new optical configuration and present preliminary results of instrument performance. Comparison with a conventional orthogonal optical geometry is made using fluorescent microspheres, chicken red blood cells and chinchilla sperm.

When conducting flow cytometric data analyses or cell sorting which results in classification of cells into two or more subpopulations, it is difficult to know which method is providing the best method and further classified using other methods.In this work we describe high-speed cell classification methods suitable for real-time data classification or cell sorting. Real-time generation of statistical classifiers and methods for comparing different classification methods by ROC analysis are also discussed. Multiparameter data mixtures of human MCF-7 breast cancer cells and human bone marrow wee analyzed by several cell classification systems including cluster analyses, principal components and discriminant function analysis. True classifier tags, implemented as additional correlated listmode parameters not used for these analyses, were used to uniquely identify each cell type and to compare classifier results. The performance of classifier systems was also assessed using ROC analysis. Preliminary results are discussed in terms of the advantages/disadvantages and problems/pitfalls of each method.

The International Society for Analytical Cytology, ISAC, has developed a flow cytometry standard (FCS) to permit data interchange, ISAC will soon replace FCS 2.0 with FCS 3.0. Unfortunately, the proposed FCS 3.0 is still fraught with problems, which are of sufficient magnitude as to warrant its early replacement. The most reasonable replacement is as a supplement to the digital imaging and communications in medicine, DICOM 3.0, standard. The recent digital microscopy extension of DICOM can be extended and modified to include flow cytometry data. DICOM includes: image graphics objects, specifications for describing: studies, reports, the acquisition of the data and the individuals involved, physician, patient, etc. Storing the present FCS data in a database, which has already been accomplished with the QC tracker software, will facilitate the transition of FCS to DICOM.

We have built several flow cytometers (FCMs) which incorporate the principle of light collection using a flow- stream waveguide (FSW). The smallest and most rugged version uses a diode laser emitting 820-nm light and collects perpendicular light scatter (PLS) with the FSW technique and collects forward light scatter (FLS) with a simple external lens. Both PLS and FLS detectors are silicon diodes with integral preamps; no fluorescence is collected. Two other units have used argon-ion lasers and a dichroic filter to separate PLS and fluorescence, and our studies have examined the factors which limit the performance of FSW light collection for low-level-fluorescence applications. In one case we have used a main-frame laser with line-tunable output and two cylindrical lenses for focusing onto the flow stream. This arrangement has allowed us to detect 93-nm yellow-green beads from Molecular Probes, Inc. and 140-nm green beads from Duke Scientific, Inc. A more recent 'luggable' version incorporates an air-cooled ion laser and a simple spherical lens for focusing the laser onto the flow stream. This FCM was able to detect the 93-nm beads from Molecular Probes. In all cases a flow stream in air is used as an optical waveguide that traps and transports light to one or more detectors.

There is a growing interest for flow and image cytometry analytes permitting the simultaneous discrimination of 6-10 fluorochromes. We have derived a strategy to optimize the factors affecting the sensitivity and specificity of multiple immunofluorescence analysis. Following a spectrophotometric analysis of 14 fluorochromes conjugated to streptavidin (SA), a set of 7 spectrally separable SA- dyes and appropriate filter combinations were selected for evaluation in image cytometry. The SA-dyes were bound to latex particles labeled with biotinilated mIgG1 and the emissions of all fluorochromes detected by each filter combination were measured. The resulting crosstalk matrix served as the basic tool for final selection of dyes, design of optimal trichroic beamsplitter and filter combination, modulation of illumination and mathematical correction of residual spectral overlap. Using this strategy we demonstrated that latex-bound SA conjugates of Cascade Blue, Lucifer Yellow, FITC, R-PE, Red613, PerCP and APC could be discriminated. More recently we extended the applicability of the technique by analyzing blood cells bound to glass slides. The same field could be initially measured for autofluorescence and non-specific IgG binding and then remeasured for specific binding of lineage markers. The ability to use paired measurements of background and total fluorescence is a significant advantage of image over flow cytometry.

A special device able to perform fast mixing and stirring of samples with different reagents was placed inside the flow chamber of a flow cytometer. This mixing device is coupled with steppe-motor driven syringes which are able to inject desired volumes of sample and reagents under computer control. The fast mixing is performed by means of a small magnetic rod vibrating in alternating magnetic field. The device may work both in continuous and stepwise manner delivering defined volumes of sample and reagent at set time intervals. Internal volume of the mixing chamber is about 0.2 (mu) l. The frequency of stirring can vary up to 1 kHz thus providing different modes: mild mixing, breaking of cell aggregates, and cell-membrane rupture. This device was used for studies of DNA content distribution in cell cycle analysis, binding kinetics of DNA-specific dyes and for analysis of chromosome sets from prestained mitotic cells It is shown that the breaking of aggregates is essential for correct estimation of the S-phase. The most significant effects are observed in the case of activated lymphocytes and for the analysis of samples from solid tumors. Breaking of cell aggregates has been achieved by a simplified device placed just prior to the flow chamber.

A new mixing and stirring device (MSD) was used to perform flow karyotype analysis of single human mitotic chromosomes analyzed so as to maintain the identity of chromosomes derived from the same cell. An improved method for cell preparation and intracellular staining of chromosomes was developed. The method includes enzyme treatment, incubation with saponin and separation of prestained cells from debris on a sucrose gradient. Mitotic cells are injected one by one in the MSD which is located inside the flow chamber where cells are ruptured, thereby releasing chromosomes. The set of chromosomes proceeds to flow in single file fashion to the point of analysis. The device works in a stepwise manner. The concentration of cells in the sample must be kept low to ensure that only one cell at a time enters the breaking chamber. Time-gated accumulation of data in listmode files makes it possible to separate chromosome sets comprising of single cells. The software that was developed classifies chromosome sets according to different criteria: total number of chromosomes, overall DNA content in the set, and the number of chromosomes of certain types. This approach combines the high performance of flow cytometry with the advantages of image analysis. Examples obtained with different human cell lines are presented.

Fluorescence lifetime measurements provide insights int eh dynamic and structural properties of dyes and their micro- environment. The implementation of fluorescence lifetime measurements in flow cytometric systems allows to monitor large cell and particle populations with high statistical significance. In our system, a modulated laser beam is used for excitation and the phase shift of the fluorescence signal recorded with a fast computer controlled digital oscilloscope is processed digitally to determine the phase shift with respect to a reference beam by fast fourier transform. Total fluorescence intensity as well as other parameters can be determined simultaneously from the same fluorescence signal. We use the epi-illumination design to allow the use of high numerical apertures to collect as much light as possible to ensure detection of even weak fluorescence. Data storage and processing is done comparable to slit-scan flow cytometric data using data analysis system. The results are stored, displayed, combined with other parameters and analyzed as normal listmode data. In our report we discuss carefully the signal to noise ratio for analog and digital processed lifetime signals to evaluate the theoretical minimum fluorescence intensity for lifetime measurements. Applications to be presented include DNA staining, parameters of cell functions as well as different applications in non-mammalian cells such as algae.

A phase-sensitive flow cytometer has ben developed to measure fluorescence emission signals from particles and cells labeled with fluorophores based on excited-state lifetimes and to quantify fluorescence lifetimes directly as a parameter in real time. This instrument combines flow cytometry (FCM) and frequency-domain, fluorescence lifetime spectroscopy measurement principles to provide unique features for making analytical cytology measurements in flow, while maintaining conventional FCM measurement capabilities. In this study we present the underlying theory of the phase-sensitive detector, along with the principles of phase-sensitive detection with respect to resolving fluorescence emission signal from background interferences caused by (1) Rayleigh scatter, (2) unbound/free dye, and (3) low-level intrinsic cellular autofluorescence, all based on differences in phase-shifts of the signals input to a phase-sensitive detector with respect to a fixed reference signal. Investigative studies directed at detecting and eliminating particle- and cell-associated and steady-state background interferences in fluorescence measurements are underway. Examples to illustrate the application of this new technology to biological problems encountered in flow cytometry applications will be presented.

For certain applications in microscopy, mercury vapor short arc lamps are utilized as UV-light sources because of their high intensity and their road spectrum. Unfortunately, they are also very unstable. Especially for single wavelength fluorescence image cytometry, there is a need for a stable, high intensity light source. Substantially improved stability was achieved using optical feedback and fiberoptic scrambling. The system uses a photodiode to monitor the light intensity, and feeds the readout back to a controller. The controller compares this readout to a preset reference voltage and adjusts the lamp supply current accordingly. The optical fiber scrambles the light to correct the effects of arc wander. Preliminary results of performance tests of this system show a coefficient of variation (CV) of less than 0.1 percent over 20 hours at a sample frequency of 30 Hz. This CV is a factor of 30 better than a conventional current stabilized mercy vapor short arc lamp. Scrambled optical feedback is a necessary addition for systems with mercury short arc lamps, especially for image fluorometry applications.

Recent improvements in analytical cytology instrumentation require the ultimate in performance from photosensors. Increases in sensitivity, extension of the spectral sensitivity range into the red, improvement of the response time and linear/area sensing are needed. This paper will introduce various photosensors recently developed by Hamamatsu. They will be sorted into groups based on performance or functional criteria as follows: (1) Photosensors: 1.1 Spectral response; We will present the variations in photoemissive materials for photomultiplier tubes (PMT) and solid state devices (SSD). 1.2 High speed; A list of high speed photosensors will be presented. In addition, we will introduce a compact module for phase modulation which is suitable for fluorescence analysis at a fast time range. 1.3 Gating; We will introduce a fast PMT gating module suitable for fluorescence measurement. The operating principle and characteristics are discussed. (2) Image sensors: a newly developed image sensor for spectroscopy will be introduced. They are Si linear image sensors for spectroscopy, InGaAs C-MOS linear image sensors for spectroscopy and full frame transfer (FFT) CCD's for low light imaging. We will discuss the outline and the characteristics of these sensors.

The use of charge coupled devices (CCDs) as non-imaging sensors in flow cytometric systems to replace the classical photomultplier tubes (PMTs) is very advantageous: the quantum efficiency of the CCDs is about 5 to 10 times higher as for PMTs, the charge storage capability of CCDs avoids analogue processing of the fluorescence signals, the dynamic range is up to 18 bits and the fluorescence intensity at different wavelengths can be recorded on the same chip. In this report a full frame CCD imager is used in a thermoelectrically cooled environment. The output signal for the CCD is digitized with a 12-bit ADC and the data are sorted as list-mode data typically used in flow cytometric work. The performance of the system is demonstrated with DNA staining of mammalian cells with acridine-orange, propidium iodide and ethidium bromide. DNA histograms comparable with standard flow cytometry are recorded. From the same data set pulse-widths histograms can be processed and used for doublet discrimination. The high quantum efficiency of the CCD sensors is of special interest for fluorescing dyes in the dark red or near IR wavelength range.

A newly developed 60W xenon flash lamp, L6604 and L6605, achieves the goals of longer operating life, higher output, and improved light stability. It operates at 2 Joules per flash input energy with approximately a 4 microsecond flash duration. The stability achieved is 2-3 percent peak-to-peak during a lifetime of 5 X 10e7 flashes, which is almost double that of conventional xenon flash lamps. This newly developed xenon flashlamp should serve as an excellent light source for analytical cytology and other fluorescence instruments. It can function as a high output, stable excitation light source for conventional fluorescence or delayed luminescence with a CCD. Besides providing powerful and stable illumination for absorption analysis of cells on slides, this lamp eliminates the optical artifacts associated with vibration of the stage which often limit throughput. This paper will describe in detail performance improvements obtained from this newly developed xenon flash lamp.

The description of an analog autofocus circuit design, developed for a scanning microscope, is presented. The analog circuit measures the degree of focus directly from the video signal of the microscope CCD camera. The circuit then returns an index to the host computer for the purpose of adjusting the position of the objective lens to bring the object in focus. Best focus is found by comparing indices at several different vertical positions. The criterion adopted for determining the degree of focus is derived from the energy distribution of the video signal spectrum. The high frequency energy of the video spectrum is a maximum at best focus and as the optics defocus, the distribution shifts to lower frequencies. A previous digital autofocus implementation required dedicated real time image processing hardware. Real time autofocus is an order of magnitude less expensive with the analog circuitry described here than it was with the dedicated digital pipeline image processing. The characteristics of the analog design and its performance are compared to that of the previous digital implementation.

An important point in the automated evaluation of molecular cytogenetic preparations is focusing the specimen before taking images. The in-focus position can vary for different positions on the slide, thus every new field of view has to be refocused. Another problem is that objects in one field of view can lie in different planes with the effect that one object is out-of-focus while the other one is in-focus. We want to investigate the importance of focus with regard to measurements like object intensity. Diffraction wave theory leads to a formula, which can be used to determine the depth-of-focus (DOF), the tolerance in the in-focus position where the image is still sharp. However practice shows that it is possible to get good measurement results even if the specimen is not imaged perfectly.the relation between the theoretical and practical DOF is analyzed. The objects under investigation are either 2D or 3D and are imaged in absorption and fluorescence mode. It appears that the DOF based on measurements can be 5 to 10 times bigger than a DOF based on wave theory with only little loss of accuracy.

Raster scanning a microscope slide to obtain morphometric and fluorometric parameters of cells requires a method for tracking the focus positions. The vertical position of each field is unpredictable because of the irregularity of glass surfaces, the flexibility of thin coverslips, the mechanical instability of microscope and the varying thickness of the tissue layers. The conventional method for vertical tracking is autofocus. For autofocus, the best focus is calculated from measures of sharpness computed on a set of images acquired at each of several vertical test positions. The focus is then set to this position for acquisition of a final image. The vertical position could also be tracked by voxel projection, with the set of test images at a microscope field treated as a single 3D image of voxels. A 2D projection is created from a 3D image by retaining only the in-focus voxels. For fluorescence confocal microscopy, a projection is usually formed by selecting the maximum intensity voxels. Another technique would be to use the maximum absolute value of the highpass filtered image. The highpass projection may have advantages for fluorescence images and, unlike the maximum projection, would also be applicable to transmission microscopy. The advantage of highpass projection is that the final image is a composite of the most sharply focused voxels. The advantages and disadvantages of these vertical positioning techniques are compared with images of DAPI-stained cell nuclei.

Interference- and phase-contrast microscopy are techniques developed to visualize transparent objects without the incorporation of stains, allowing observations on living cells. The development of CCD cameras and image analysis technology in the last decades permits quantitative analysis of interferometric data, obtained by interference microscopy. A method is presented to determine the optical pathlength at each position in digitized microscopic images. Unstained cells and artificial test objects were observed under a standard, commercially available, Nomarski differential interference contrast (DIC) microscope, using monochromatic light. A cooled CCD camera was used to record images. Image processing software was used to convert the measured intensities to phase differences. A wiener filter based on the typical lateral shift introduced by the DIC microscope, was applied to reconstruct the original optical pathlength distribution. This system, based upon a conventional DIC microscope, rather than upon a purpose build interferometric scanning system, allows easier determination of optical pathlength differences. After image acquisition and determination of optical pathlength distribution, analysis of cell parameters can be performed using standard image processing software. Dry mass- or DNA content, for instance, can be estimated by integrating the optical pathlength over the cellular surface.

Many biological objects, include blood cells, are optically transparent. They do not absorb light, and only change optical path length by variations of object's thickness or its refractive index variations. For visualization of phase information and obtaining quantitative properties of cells we use phase-shift interferometry. Cells are fixed by glutaraldehyde and mounted in immersion liquid for reduction the influences of refraction. Characteristic peculiarity of interferometric microscope are two channels of optical scheme: object and reference. Blood cell's interferogram contain the information about space distribution refractive index. For its reconstruction we use phase-shift method. This method includes record of four interferograms used different values of phase-shift of the reference wave. Many blood cells interferogram are obtained. The shape, size and density of cells are measured.

Optical methods of monitoring blood analytes are attractive to the medical community as alternatives to traditional chemical assays. However, the calibration process is complicated by many factors, such as overlapping spectral bands from different analytes, background noise, and interpatient variability, making the choice of measurement technique crucial. In our studies, we use near-IR Raman spectroscopy in order combine the advantages of deep penetration depths and sharp, distinct peaks. Our system consists of an 830 nm diode laser, fiber-based light delivery and collection, an f/1.8 holographic spectrograph, and a liquid nitrogen-cooled CCD detector. Blood samples from a nearby hospital are placed in cuvettes and stirred throughout the measurements to reduce heating effects. Partial least squares calibration is performed using the Raman spectra and the reference analyte concentrations from the hospital. Prediction uncertainties are calculated by cross-validation using the leave-one-out method. Using scans of fifteen minutes, we are able to extract the concentration of total protein and other analytes form serum samples with clinically acceptable levels of uncertainty. Whole blood studies are currently underway.

We investigated the impact of the scattering phase function approximation on the optical properties of whole human blood determined from integrating sphere measurements using an inverse Monte Carlo technique. The diffuse reflectance Rd and the total transmittance Tt ((lambda) equals 633nm) of the whole blood samples were measured with a double integrating sphere equipment. The experimental scattering phase functions of the highly diluted blood samples were measured with a goniophotometer. We approximated the experimental scattering phase function with Mie, Gegenbauer kernel (GKPF), and Henyey-Green (HGPF) phase functions to pre-set the anisotropy factor (mu) for the inverse problem. We have employed HGPF, GKPF, and MPF approximations in the inverse Monte Carlo procedure to derive the absorption coefficient (mu) _a and the scattering coefficient (mu) _s. The results show significant difference in the final estimates of (mu) _s. 12

A sample of chylomicrons from human lymph was characterized using dynamic light scattering measurements. The stability of the sample was checked by carrying out series measurements at 3 different days, and was found to be very high during this period. The size distribution was determined by means of an extended version of the algorithm CONTIN that allowed normalization errors to be taken into account. We applied here a special evaluation scheme, where the data were corrected for these errors before final analysis. In addition to the intensity weighted size distribution obtained from this method we derived the size distributions of number and mass, utilizing the scattering amplitude functions of Mie. For these calculations the intensity-weighted size distribution was refined using an interpolation scheme to reduce problems associated with the use of mean values of the scattering amplitude functions for the intervals of the original size grid. The results showed that the chylomicron sample contained two classes of particles with intensity-weighted mean diameters of 107 nm and 320 nm. Number and mass distributions derived for the two major peaks indicated that practically all particles were small ones, but also that the few remaining large ones were carrying about 10 percent of the total mass.