Valeri P. Maltsev Estimation of morphological characteristics of single particles from light scattering data in flow cytometry", Russian Chemical Bulletin, v. 43, No. 7, pp. 1115-1124 (1994) Numerical methods for solving the reverse problem of light scattering for single particles and a variety of experimental results have been reviewed. In particular, the two-dimensional Mie scattering method for the estimation of sizes and refractive indices of single particles has been developed. The method of triple two-dimensional Mie scattering has been elaborated, which extends the range of particle sizes with correct solution of the reverse light scattering problem. The flying light scattering indicatrix method, which allows the estimation of the parameters of spherical particles, is presented. The results of the use of a flow cytometer of standatd design for the analysis of milk microflora and for an AIDS immunoassay by latex agglutination are given. The design of a scanning flow cytometer is considered. The results of the measuring the light scattering from polystyrene latex particles are given ------------ Valeri P. Maltsev, Andrej V. Chernyshev , Alexander A. Doroshkin, and Erkki Soini . Light scattering and fluorescence of single particles measured by a scanning flow cytometer. Proceedings of SPIE, 2386: 199-205 (1995) A novel type of Scanning Flow Cytometer (SFC) has been developed for improving particle analysis from light scattering data. The use of the SFC and the Flying Light Scattering Indicatrix (FLSI) method allows absolute measurement of the size and refractive index of spherical particles. The separation efficiency of the single particle analysis using FLSI method is verified measuring the size distribution of standard latex particles. It is shown that particles differed in size more then 1.2 um can be separated by the FLSI method to different classes. In second part of this paper the feasibility of a Pulsed Laser Flow Cytometer (PLFC) equipped by a pulsed nitrogen laser is tested for fluorometric analysis of human lymphocytes stained with FITC-labeled monoclonal antibodies. In addition the feasibility for the fluorescence measurement in time-resolved regime of the PLFC is analyzed from measurement of photon collection efficiency of scanning optical system. ----------- A. V. Chernyshev , V. I. Prots, A. A. Doroshkin, and V. P. Maltsev Measurement of scattering properties of individual particles with a scanning flow cytometer. Applied Optics 34, 6301-6305 (1995) A hydrofocusing head with an optical cuvette has been developed for the flow cytometer to generate complete scatter patterns of single particles at scattering angles ranging from 10 to 120. The scatter signal has been measured as a function of the angle (a flying indicatrix) by the use of particle motion within a scanning system of the flow cytometer by the use of a single photomultiplier. Scattering data measured with the flow cytometer have been compared with those calculated from Mie theory for latex particles. A calculation algorithm has been used to estimate the size and the refractive index of spherical particles from the scattering data measured. ----------- V. P. Maltsev, A. V. Chernyshev , K. A. Sem'yanov, and E. Soini . Absolute real-time measurement of particle size distribution with the flying light-scattering indicatrix method Applied Optics 35, 3275-3280 (1996) The flying light-scattering indicatrix (FLSI, angular dependency of the intensity of light scattered by a moving individual particle) method, based on a scanning flow cytometer (SFC) that permits measurement of individual particle characteristics from light-scattering data, has been used for the determination of size distribution of the following particles: polystyrene latex, milk fat, and spores (Penicillium levitum, Aspergillus pseudoglaucus). The optical system of the SFC and empirical equations provided absolute sizing at the rate of 50 particles/s. Size distributions obtained with the FLSI method and a best-fit procedure using Mie Scattering theory have been compared. ---------- V. P. Maltsev and V. N. Lopatin . Parametric solution of the inverse light-scattering problem for individual spherical particles Applied Optics 36, N 24, pp. 6102-6108 (1997) We analyze the relation between spherical particle parameters (size parameter alpha and relative refractive index m') and light-scattering indicatrix parameters (fringe pitch and visibility) for alpha and m' ranging from 7 to 88 and from 1.025 to 1.2, respectively. We consider formation of the indicatrix structure under variation of size parameter alpha and phase-shift parameter ro = 2alpha(m' - 1). We show that indicatrix visibility depends basically on the phase-shift parameter whereas fringe pitch depends on the size parameter. An analysis of these dependencies allows us to determine the region in the alpha x ro map where particle parameters can be derived unambiguously from indicatrix parameters. We clarify errors in determination of particle characteristics on the basis of the approximating equations that relate particle size and phase shift to the indicatrix parameters. ---------- V P Maltsev, A V Chernyshev, K A Sem'yanov, and E Soini Absolute real-time determination of size and refractive index of individual microspheres Measurement Science and Technology, v. 8, N 9, pp. 1023-1027 (1997) The flying light scattering indicatrix (FLSI) method has been improved by employing a set of empirical equations combined with a decision tree which has been used for simultaneous real-time measurements and calculation of the size and refractive index of individual spherical particles. Empirical equations have been developed for determination of particle parameters over the range 1 um to 12 um and 1.37 to 1.60 for size and refractive index respectively. The FLSI method has been used for determination of the precision of a scanning flow cytometer for analysis of individual particles. A two-dimensional map (size & refractive index) was created for polystyrene latex and milk fat particles. ---------- A.V.Chernyshev, A.E.Soini, I.V.Surovtsev, V.P.Maltsev, and E.Soini A mathematical model of dispersion radical polymerization kinetics Journal of Polymer Science, Polym. Chem., v. 35, N 9, p. 1799 (1997) A mathematical model of the basic polymerization kinetic scheme, that involves only initiation, propagation, and termination reactions, has been developed. It was assumed that the propagation and termination rate constants are independent of the chain length. It has been shown that the model predicts the effect of autoacceleration on the formation of the insoluble polymer component. In order to demonstrate the applicability of the mathematical model for description of dispersion radical polymerization, the evolution of the size distribution of growing polystyrene particles has been measured with the Flying Light-Scattering Indicatrix method. ---------- V.P. Maltsev, A.V. Chernyshev Method and device for determination of parameters of individual microparticles US Patent Number: 5,650,847. Date of patent: Jul. 22, 1997 This invention relates to a method and device for analysing morphological parameters of microparticles. The microparticles in this context mean organic, polymeric or inorganic microparticles or aerosols or small biological objects including cells, spores and other living or non-living particles. The morphological parameters in this context mean the physical dimensions including minimum and maximum diameter and the optical refractive index. In particular, this invention provides a fast and absolute methodology for measuring the diameter and the refractive index of individual spherical particles with the size from 0.8 um to 100 um. ---------- Juhani T. Soini, Andrei V. Chernyshev, Pekka E. Hanninen, Erkki Soini, and Valeri P. Maltsev A New Design of the Flow Cuvette and Optical Set-Up for the Scanning Flow Cytometer Cytometry v. 31, N 2, pp. 78-84 (1998) We introduce a new design for the optical cuvette and a new optical lay-out for the Scanning Flow Cytometer (SFC) that permits measurement of the angular dependency of the scattered light from individual moving particles. The improved optical scheme of the SFC allows measurement of the angular scattering pattern of individual particles at polar angles from 10° to 120° with integration at alimuthal angles from 0° to 360° and with angular resolution of better than 0.5°. The performance of the SFC is demonstrated using certified polystyrene particles as reference material. The aim of this work is to develop a flow cytometer, which, by recording the entire light scattering pattern of individual biological particles, would provide more information about the particle structure; than the ordinary wide angle, forward and side scattering concepts. ---------- A.V. Chernyshev, A.E. Soini, V.P. Maltsev and E. Soini A model of complete classical treatment of dispersion radical polymerization kinetics Macromolecules 31, pp. 6455-6460 (1998) Kinetic analysis of high-conversion free radical polymerization is discussed in terms of the complete solution of the classical kinetic scheme, that involves initiation, propagation, termination and chain transfer reactions. Expressions for kinetics of concentrations of each of the species existing in the polymerization mixture are presented. In order to demonstrate the applicability of the mathematical model, monomer conversion and the formation of the insoluble polymer component in the dispersion polymerization of styrene (ST) and methyl methacrylate (MMA) in isopropanol have been measured at 700C. The values obtained for kd and kp/kt1/2, where kd, kp and kt are dissociation, propagation and termination rate constants, respectively, are in agreement with those reported in the literature. ---------- A.V. Chernyshev, A.E. Soini Free Radical Dispersion Polymerization of Styrene in a Mixture of 2-propanol and Tetrahydrofuran Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 36, 2757–2761 (1998) Free radical dispersion polymerization of styrene in a mixture of 2-propanol and tetrahydrofuran was carried out at 70°C up to high conversions. The influence of the change of the critical chain length on the evolution of the insoluble polymer component was examined. Monomer conversion and the formation of the insoluble polymer component were measured in order to test a mathematical model presented in our previous article.18 The critical polymer chain length i0 , the initiation rate constant kd , and the ratio kp /kt 1/2 , where kp and kt are propagation and termination rate constants, respectively, have been obtained and compared with those reported in the literature. ---------- A. N. Shvalov , J. T. Soini , A. V. Chernyshev , P. A. Tarasov , E. Soini , V. P. Maltsev Light-Scattering Properties of Individual Erythrocytes Applied Optics, Vol. 38, 230 - 235 (1999) We have studied the light-scattering properties of human erythrocytes both experimentally and theo-retically. In the experimental study measurements were performed with a Scanning Flow Cytometer (SFC). The SFC can measure the light-scattering pattern (indicatrix) of an individual particle over an angular range of 10-60°. We have observed polymorphism in the measured set of indicatrices. To understand the reason for this polymorphism, we have made a theoretical study of the scattering properties of erythrocytes. The Wentzel-Kramer-Brillouin approximation has been employed to calculate indicatrices of individual erythrocytes in different orientations relative to the incident light beam. The indicatrices were calculated over an angular range of 15-35°. A comparison of the experimentally measured and theoretically calculated indicatrices shows that the polymorphism is due mainly to the different orientation of the erythrocytes in the flow. The effect caused by the Poiseuille profile of the flow on an individual erythrocyte within the SFC cuvette capillary was studied theoretically by use of the Stokes approximation. Rotation of an erythrocyte was predicted by this theoretical analysis, and this prediction was further verified by comparison with experimental results. ---------- N.V.Shepelevich, V.V.Lopatin, V.P.Maltsev, and V.N.Lopatin Extrema in the light-scattering indicatrix of a homogeneous sphere Journal of Optics A: Pure and Applied Optics v. 1, N 4, 448-453, July 1999 Formation and migration of extrema in the angular dependency of the light-scattering intensity (an indicatrix), under variation of the size and the refractive index of a homogeneous sphere, have been studied analytically and numerically. Approximations of Wentzel–Kramers–Brillouin (WKB), Rayleigh–Gans–Debye (RGD), Fraunhofer diffraction, and anomalous diffraction (AD) have been considered for the relative refractive index ranging from 1.025 to 1.200. Utilization of these approximations has allowed us to reveal the generalized coordinates. The plot of extrema locations of the indicatrix calculated from the WKB approximation with these coordinates has shown a universal mechanism of formation of the indicatrix structure. We determined the upper boundary of the angular region of Fraunhofer diffraction (FD) applicability. An analysis of the extrema has shown that locations of the different minima migrate monotonically when the size of the sphere is varied. An empirical equation that relates the minimum location to the sphere size has been produced. It has been shown that the distance between minima is insensitive to variations of the relative refractive index. The disappearance of the minima with an increasing relative refractive index has been discussed. ---------- A.N.Shvalov, I.V.Surovtsev, A.V.Chernyshev, J.T.Soini, and V.P.Maltsev Particle Classification from Light Scattering with the Scanning Flow Cytometer Cytometry Volume 37, Issue 3, 1999, pp. 215-220 Background: The differential light-scattering pattern, an indicatrix, provides the most complete characterization of the optical properties of a particle. Particle classification can be performed on the basis of particle parameters retrieved from the indicatrices. This classification extends the ability of flow cytometry in particle recognition. Methods: The scanning flow cytometer (SFC) permits an acquisition of traces of light scattering signals, i.e., native SFC traces, from single particles. The acquired native SFC traces are transformed into indicatrices. The performance of the SFC in measurements of indicatrices has been demonstrated for the following particles: lymphocytes, erythrocytes, polystyrene particles, and milk-fat particles. Results: The structure and profile of the indicatrix for each particle type have been found to be unique. Classifica-tion of polystyrene particles has been performed on the basis of the map formed by particle refractive index and size. The polystyrene particles were classified using this map into different size categories ranging from 1.4–7 µm, with a size deviation of 0.07 µm. Conclusions: The method based on analysis of native SFC traces shows better performance in particle classification than the method based on the particle refractive index and size map. The classification performance of the SFC will be useful, for example, for particle sorting and particle identification, and with additional fluorescent measure-ments may have applications in multiparameter particle-based immunoassay. ---------- V.P.Maltsev Scanning flow cytometry for individual particle analysis Review of Scientific Instruments -- January 2000 -- Volume 71, Issue 1, pp. 243-255 With this work we review the development of theoretical and experimental aspects of the scanning flow cytometry ~SFC!. The optical and hydrodynamic systems of the SFC provide the measurement of fluorescence and light scattering of individual particles with a typical rate up to 500 particles/s. In addition the optical system of the SFC has the capability of individual particle analysis beyond that of an ordinary flow cytometry. The SFC measures an entire angular dependency of light scattering intensity ~flying light scattering indicatrix, FLSI! over angles ranging from 5° to 120°. The fluorescence collection efficiency of the SFC approaches 1/3 of a sphere. Moreover, the optical system of the SFC provides the measurement of fluorescence in a time-resolved mode on a microsecond time scale. The processing of the output data in light scattering is based on a parametric solution of the inverse light-scattering problem, the FLSI method. The FLSI method allows the determination of size and refractive index of spherically modeled particles over a range of diameters from 0.9 to 15 mm and a range of refractive indexes from 1.37 to 1.60. The performance of the SFC in different applications has been demonstrated. ---------- Alexander N. Shvalov, Juhani T. Soini, Ivan V. Surovtsev, Galina V. Kochneva, Galina F. Sivolobova, Alexander K. Petrov, Valeri P. Maltsev Individual Escherichia coli cells studied from light scattering with the scanning flow cytometer Cytometry Volume 41, Issue 1, 2000, pp. 41-45 Background: Flow cytometry is a powerful tool for the analysis of individual particles in a flow. Differential light scattering (an indicatrix) was used for many years to obtain morphologic information about microorganisms. The indicatrices play the same role for individual particle recognition as a spectrum for substance characterization. We combined two techniques to analyze the indicatrix of the cells for the purpose of developing a database of light-scattering functions of cells. Methods: The scanning flow cytometer (SFC) allows the measurement of the entire indicatrix of individual parti-cles at polar angles ranging from 5° to 100°. In this work, light-scattering properties of Escherichia coli have been studied both experimentally and theoretically with the SFC and the T-matrix method, respectively. The T-matrix method was used because of the nonspherical shape of E. coli cells, which were modeled by a prolate spheroid. Results: The indicatrices of E. coli cells were stimulated with T-matrix method at polar angles ranging from 10° to 60°. The absolute cross-section of light scattering of E. coli has been determined comparing the cross section of poly-styrene particles modeled by a homogeneous sphere. The E. coli indicatrices were compared for logarithmic and stationary phases of cell growth. Conclusions: The indicatrices of E. coli were reproduc-ible and could be used for identification of these cells in biologic suspensions. The angular location of the indica-trix minimum can be used in separation of cells in loga-rithmic and stationary phases. To use effectively the indi-catrices for that purpose, the light-scattering properties of other microorganisms have to be studied. ---------- Ivan V. Surovtsev, Ivan A. Razumov, Vyacheslav M. Nekrasov, Alexander N. Shvalov, Juhani T. Soini, Valeri P. Maltsev, Alexander K. Petrov, Valeri B. Loktev, Andrei V. Chernyshev Mathematical Modeling the Kinetics of Cell Distribution in the Process of Ligand-Receptor Binding Journal of Theoretical Biology, Vol. 206, No. 3, pp. 407-417 (October 2000) A statistical approach is presented to model the kinetics of cell distribution in the process of ligand}receptor binding on cell surfaces. The approach takes into account the variation of the amount of receptors on cells assuming the homogeneity of monovalent binding sites and ligand molecules. The analytical expressions for the kinetics of cell distribution have been derived in the reaction-limited approximation. In order to demonstrate the applicability of the mathematical model, the kinetics of binding the rabbit, anti-mouse IgG with Ig-receptors of the murine hybridoma cells has been measured. Anti-mouse IgG was labeled with #uorescein isothiocyanate (FITC). The kinetics of cell distribution on ligand}receptor complexes was observed during the reaction process by real-time measuring of the #uorescence and light- scattering traces of individual cells with the scanning #ow cytometer. The experimental data were "tted by the mathematical model in order to obtain the binding rate constant and the initial cell distribution on the amount of receptors. ---------- Konstantin A Sem’yanov, Pit A. Tarasov, Juhani T. Soini, Alexander K. Petrov, and Valeri P. Maltsev Calibration free method to determine the size and hemoglobin concentration of individual red blood cells from light scattering Applied Optics v. 39, 11, pp. 5884 – 5889 (2000) At present, hemoglobin concentration and volume of an erythrocyte can be determined from intensities of light scattered by an individual cell at fixed angular intervals. This method is used in modern hemoglobin analyzers but it requires calibration of optical and electronic units by certified particles of known size and refractive index. In this study we show a method which is based on the parametric solution of inverse light-scattering problem and which does not require calibration procedure. The method is based on the use of parameters of the entire angular light-scattering pattern, here called an indicatrix. These parameters do not depend on absolute intensity of light scattering. The indicatrix parameters form approximating equations that relate these parameters to size and phase-shift parameters of the particle. Applicability of the method is demonstrated with measurement of indicatrices of individual sphered erythrocytes. The indicatrices of the individual erythrocytes were measured with a scanning flow cytometer at angular range from 15 to 55 degrees. The volume and the hemoglobin concentration have been calculated using the developed method and by fitting the experimental indicatrices to the indicatrices calculated from the Mie theory. ---------- Meas. Sci. Technol. 19 (2008) 015408 The scanning flow cytometer modified for measurement of two-dimensional light-scattering pattern of individual particles Gleb V Dyatlov1,3, Konstantin V Gilev1, Konstantin A Semyanov2 and Valeri P Maltsev We theoretically consider a new approach for measurement of the two-dimensional light-scattering patterns (2D LSP) of individual particles (for example, blood cells). Unlike the original optical scheme of the scanning flow cytometer that integrates scattering intensity over the azimuth angle, the new scheme allows us to measure the 2D LSP. The approach assumes measurement of the integral distribution of intensity on the fixed plane with subsequent reconstruction of the pattern via solving a first-kind integral equation. The last problem is ill-posed and we solve this equation by the standard regularization method. Error sources of the new approach are discussed from a comparison of the initial and reconstructed 2D LSPs for non-spherical particles. ---------- Maxim A. Yurkin, Konstantin A. Semyanov, Valeri P. Maltsev, and Alfons G. Hoekstra Discrimination of granulocyte subtypes from light scattering: theoretical analysis using a granulated sphere model Optics Express, Vol. 15, Issue 25, pp. 16561-16580 (2007) Abstract: We perform extensive simulations of light scattering by a granulated sphere in the size and refractive index range of human granulated leucocytes using the discrete dipole approximation. We calculate total and depolarized side scattering signals as a function of the size and refractive indices of cell and granules, and the granule volume fraction. Using typical parameters derived from the literature data on granulocyte morphology, we show that differences between experimentally measured signals of two granulocyte subtypes can be explained solely by the difference in their granule sizes. Moreover, the calculated depolarization ratio quantitatively agrees with experimental results. We also use the Rayleigh-Debye-Gans approximation and its second order extension to derive analytical expressions for side scattering signals. These expressions qualitatively describe the scaling of signals with varying model parameters obtained by rigorous simulations, and even lead to quantitative agreement in some cases. Finally, we show and discuss the dependence of extinction efficiency and asymmetry parameter on size and volume fraction of granules. ---------- Yurkin M.A., Hoekstra A.G. The discrete dipole approximation: an overview and recent developments Journal of Quantitative Spectroscopy & Radiative Transfer, v.106(1-3), pp. 558-589 (2007). We present a review of the discrete dipole approximation (DDA), which is a general method to simulate light scattering by arbitrarily shaped particles. We put the method in historical context and discuss recent developments, taking the viewpoint of a general framework based on the integral equations for the electric field. We review both the theory of the DDA and its numerical aspects, the latter being of critical importance for any practical application of the method. Finally, the position of the DDA among other methods of light scattering simulation is shown and possible future developments are discussed. ---------- M. A. Yurkin, V. P. Maltsev, A. G. Hoekstra The discrete dipole approximation for simulation of light scattering by particles much larger than the wavelength Journal of Quantitative Spectroscopy & Radiative Transfer 106, 546-557 (2007) In this paper we investigate the capabilities of the discrete dipole approximation (DDA) to simulate scattering from particles that are much larger than the wavelength of the incident light, and describe an optimized publicly available DDA computer program that processes the large number of dipoles required for such simulations. Numerical simulations of light scattering by spheres with size parameters x up to 160 and 40 for refractive index m ? 1:05 and 2, respectively, are presented and compared with exact results of the Mie theory. Errors of both integral and angle-resolved scattering quantities generally increase with m and show no systematic dependence on x: Computational times increase steeply with both x and m, reaching values of more than 2 weeks on a cluster of 64 processors. The main distinctive feature of the computer program is the ability to parallelize a single DDA simulation over a cluster of computers, which allows it to simulate light scattering by very large particles, like the ones that are considered in this paper. Current limitations and possible ways for improvement are discussed. ---------- Tarasov P.A., Yurkin M.A., Avrorov P.A., Semyanov K.A., Hoekstra A.G., Maltsev V.P. Optics of erythrocytes Optics of Biological Particles, ed. Hoekstra A.G., Maltsev V.P., Videen G., Springer, London, pp. 231-246 (2006) We present optical methods to study erythrocytes and consider selected models to compute their light scattering. ---------- Semyanov K.A., Zharinov A.E., Tarasov P.A., Yurkin M.A., Skribunov I.G., van Bockstaele D.R., Maltsev V.P. Optics of leucocytes Optics of Biological Particles, ed. Hoekstra A.G., Maltsev V.P., Videen G., Springer, London, pp. 253-264 (2006) Optical methods to study neutrophils, eosinophils, basophils, lymphocytes, and monocytes are reviewed. Recent applications of scanning flow cytometry to characterize lymphocytes and monocytes is presented. ---------- Irina Kolesnikova, Sergey Potapov, Peter Tarasov, Konstantin Semyanov, and Valeri Maltsev Optics of platelets Optics of Biological Particles, ed. Hoekstra A.G., Maltsev V.P., Videen G., Springer, London, pp. 247-252 (2006) Optical methods to study platelets with recent application to scanning flow cytometry are presented. ---------- M.A. Yurkin, A.G. Hoekstra, V.P. Maltsev Convergence of the discrete dipole approximation. II. An extrapolation technique to increase the accuracy Journal of the Optical Society of America A, 23, pp. 2592-2601 (2006) We propose an extrapolation technique that allows accuracy improvement of discrete dipole approximation computations. The performance of this technique was studied empirically on the basis of extensive simulations for five test cases using many different discretizations. The quality of the extrapolation improves with refining discretization, reaching extraordinary performance especially for cubically shaped particles. A 2-order-ofmagnitude decrease of error is demonstrated. We also propose estimates of the extrapolation error, which are proven to be reliable. Finally, we propose a simple method to directly separate shape and discretization errors and illustrate this for one test case. ---------- M.A. Yurkin, A.G. Hoekstra, V.P. Maltsev Convergence of the discrete dipole approximation. I. Theoretical analysis Journal of the Optical Society of America A, 23, pp. 2578-2591 (2006) We perform a rigorous theoretical convergence analysis of the discrete dipole approximation (DDA). We prove that errors in any measured quantity are bounded by a sum of a linear term and a quadratic term in the size of a dipole d when the latter is in the range of DDA applicability. Moreover, the linear term is significantly smaller for cubically than for noncubically shaped scatterers. Therefore, for small d, errors for cubically shaped particles are much smaller than for noncubically shaped ones. The relative importance of the linear term decreases with increasing size; hence convergence of DDA for large enough scatterers is quadratic in the common range of d. Extensive numerical simulations are carried out for a wide range of d. Finally, we discuss a number of new developments in DDA and their consequences for convergence. ---------- F. Barnaba, L. Fiorania, A. Palucci, P. Tarasov First characterization of marine particles by laser scanning flow cytometry Journal of Quantitative Spectroscopy & Radiative Transfer 102 (2006) 11–17 A new laser scanning flow cytometer (CLASS) has been developed at ENEA to characterize single marine particles. The first setup of the instrument allowing the detection of the particle light scattering over a wide angular range is presented. A test of CLASS employing polystyrene microspheres is described. Eventually, the first measurements of biological particles (Penicillium Italicum conidia) and marine phytoplankton (Synechocystis cells) are reported. ---------- I.V. Kolesnikova, S.V. Potapov, M.A. Yurkin, A.G. Hoekstra, V.P. Maltsev, K.A. Semyanov Determination of volume, shape and refractive index of individual blood platelets Journal of Quantitative Spectroscopy & Radiative Transfer 102 (2006) 37-45 Light scattering patterns (LSP) of blood platelets were theoretically and experimentally analyzed. We used spicular spheroids as a model for the platelets with pseudopodia. The discrete dipole approximation was employed to simulate light scattering from an individual spicular spheroid constructed from a homogeneous oblate spheroid and 14 rectilinear parallelepipeds rising from the cell centre. These parallelepipeds have a weak effect on the LSP over the measured angular range. Therefore, a homogeneous oblate spheroid was taken as a simplified optical model for platelets. Using the T-matrix method, we computed the LSP over a range of volumes, aspect ratios and refractive indices. Measured LSPs of individual platelets were compared one by one with the theoretical set and the best fit was taken to characterize the measured platelets, resulting in distributions of volume, aspect ratio and refractive index. ---------- Vladimir V. Berdnik, Konstantin Gilev, Alexander Shvalov, Valeri Maltsev, Valery A. Loiko Characterization of spherical particles using high-order neural networks and scanning flow cytometry Journal of Quantitative Spectroscopy & Radiative Transfer 102 (2006) 62–72 We retrieve the radius R, real n and imaginary k parts of the refractive index of homogeneous spherical particles using angular distribution of the light-scattering intensity. To solve the inverse light-scattering problem we use a high-order neural-network technique. The effect of network parameters on optimization is examined. The technique is evaluated for noise-corrupted input data at 0.6 mmoRo10.6 mm, 1.02ono1.38, and 0oko0.03. The errors of retrieval for nonabsorbing particles do not exceed 0.05 mm for radius and 0.015 for refractive index. The experimental verification is fulfilled by experimental data retrieved by means of a scanning flow cytometer. The light-scattering profiles of polystyrene beads and spherized red blood cells are processed with the high-order neural networks and a non-linear regression at Mie theory. The parameters retrieved by the high-order neural networks correlate well with the parameters retrieved by the least-square method. ---------- Alexey Zharinov, Peter Tarasov, Alexander Shvalov, Konstantin Semyanov, Dirk R. van Bockstaele, Valeri Maltsev A study of light scattering of mononuclear blood cells with scanning flow cytometry Journal of Quantitative Spectroscopy & Radiative Transfer 102 (2006) 121–128 This study describes the measurement of light scattering of human mononuclear blood cells, the development of an appropriate optical model for those cells, and solution of the inverse light-scattering problem. The angular dependency of light-scattering intensity of mononuclear blood cells was experimentally measured by means of scanning flow cytometry. A sphere consisting of several concentric homogeneous layers with different refractive indices was tested as an optical model for mononuclear blood cells. A five-layer model has given the best agreement between experimental and theoretical lightscattering profiles. The inverse light-scattering problem was solved for a five-layer model with an optimization procedure that allows one to retrieve cell parameters: cell size relates to the outer diameter of the fifth layer; size of the nucleus relates to the outer diameter of the third layer. Mean values of cell size, nuclear size, refractive indices of nucleus and cellular cytoplasm were determined for blood monocytes and lymphocytes. ---------- Yurkin M.A., Semyanov K.A., Tarasov P.A., Chernyshev A.V., Hoekstra A.G., and V. P. Maltsev Experimental and theoretical study of light scattering by individual mature red blood cells by use of scanning flow cytometry and a discrete dipole approximation Applied Optics v. 44, pp. 5249 - 5256 (2005) Elastic light scattering by mature red blood cells (RBCs) was theoretically and experimentally analyzed by use of the discrete dipole approximation (DDA) and scanning flow cytometry (SFC), respectively. SFC permits measurement of the angular dependence of the light-scattering intensity (indicatrix) of single particles. A mature RBC is modeled as a biconcave disk in DDA simulations of light scattering. We have studied the effect of RBC orientation related to the direction of the light incident upon the indicatrix. Numerical calculations of indicatrices for several axis ratios and volumes of RBC have been carried out. Comparison of the simulated indicatrices and indicatrices measured by SFC showed good agreement, validating the biconcave disk model for a mature RBC. We simulated the light-scattering output signals from the SFC with the DDA for RBCs modeled as a disk–sphere and as an oblate spheroid. The biconcave disk, the disk–sphere, and the oblate spheroid models have been compared for two orientations, i.e., face-on and rim-on incidence, relative to the direction of the incident beam. Only the oblate spheroid model for rim-on incidence gives results similar to those of the rigorous biconcave disk model. ---------- K. A. Semyanov, P. A. Tarasov, A. E. Zharinov, A. V. Chernyshev, A. G. Hoekstra, and V. P. Maltsev Single-particle sizing from light scattering by spectral decomposition Applied Optics v. 43, pp. 5110 - 5115 (2004) A Fourier transform was applied to size an individual spherical particle from an angular light-scattering pattern. The position of the peak in the amplitude spectrum has a strong correlation with the particle size. A linear equation retrieved from regression analysis of theoretically simulated patterns provides a relation between the particle size and the location of the amplitude spectrum’s peak. The equation can be successfully applied to characterize particles of size parameters that range from 8 to 180 corresponding to particle sizes that range from 1.2 to 27.2 m at a wavelength of 0.633 m. The precision of particle sizing depends on the refractive index and reaches a value of 60 nm within refractive-index region from 1.35 to 1.70. We have analyzed four samples of polystyrene microspheres with mean diameters of 1.9, 2.6, 3.0, and 4.2 m and a sample of isovolumetrically sphered erythrocytes with a scanning flow cytometer to compare the accuracy of our new method with that of others. ---------- Konstantin Sem’yanov and Valeri P. Maltsev Analysis of Sub-Micron Spherical Particles using Scanning Flow Cytometry Particles and Particles Systems Characterization Volume 17, Issue 5-6, 2000. Pages: 225-229 Determination of individual sub-micron particle size and refractive index has been performed with a parametric solution of inverse light-scattering problem. The parametric solution is based on approximating equations that relate the particle characteristics to the parameters of the indicatrix (an angular dependency of light-scattering intensity). The approximation equations allow real-time calculation of particle size and refractive index ranging from 0.5 ?m to 1.5 ?m and from 1.37 to 1.69, respectively. An applicability of the parametric solution has been demonstrated with analysis of the polystyrene particles which indicatrices were measured with the Scanning Flow Cytometer. We have measured the probes with monodisperse polystyrene particles with diameters of 0.6 ?m and 0.83 ?m. The measured indicatrices were processed with the parametric solution and best-fit regression to the Mie theory. The determined particle parameters were in a good agreement with specifications. ---------- Andrey V. Chernyshev, Peter A. Tarasov, Konstantin A. Semianov, Vyacheslav M. Nekrasov, Alfons G. Hoekstra, Valeri P. Maltsev Erythrocyte lysis in isotonic solution of ammonium chloride: Theoretical modeling and experimental verification Journal of Theoretical Biology 251 (2008) 93–107 A mathematical model of erythrocyte lysis in isotonic solution of ammonium chloride is presented in frames of a statistical approach. The model is used to evaluate several parameters of mature erythrocytes (volume, surface area, hemoglobin concentration, number of anionic exchangers on membrane, elasticity and critical tension of membrane) through their sphering and lysis measured by a scanning flow cytometer (SFC). SFC allows measuring the light-scattering pattern (indicatrix) of an individual cell over the angular range from 101 to 601. Comparison of the experimentally measured and theoretically calculated light scattering patterns allows discrimination of spherical from non-spherical erythrocytes and evaluation of volume and hemoglobin concentration for individual spherical cells. Three different processes were applied for erythrocytes sphering: (1) colloid osmotic lysis in isotonic solution of ammonium chloride, (2) isovolumetric sphering in the presence of sodium dodecyl sulphate and albumin in neutrally buffered isotonic saline, and (3) osmotic fragility test in hypotonic media. For the hemolysis in ammonium chloride, the evolution of distributions of sphered erythrocytes on volume and hemoglobin content was monitored in real-time experiments. The analysis of experimental data was performed in the context of a statistical approach, taking into account that parameters of erythrocytes vary from cell to cell. ---------- I.V.Surovtsev, M.A.Yurkin, A.N.Shvalov, V.M.Nekrasov, G.F.Sivolobova, A.A.Grazhdantseva, V.P.Maltsev, and A.V.Chernyshev Kinetics of the initial stage of immunoagglutionation studied with the scanning flow cytometer Colloids and Surfaces B: Biointerfaces, Volume 32, Issue 3, pp. 245-255, 2003 The use of a scanning flow cytometer (SFC) to study the evolution of monomers, dimers and higher multimers of latex particles at the initial stage of the immunoagglutination is described. The SFC can measure the light-scattering pattern (indicatrix) of an individual particle over an angular range of 10/608. A comparison of the experimentally measured and theoretically calculated indicatrices allows one to discriminate different types of latex particles (i.e. monomers, dimers, etc.) and, therefore, to study the evolution of immunoagglutination process. Validity of the approach was verified by simultaneous measurements of light-scattering patterns and fluorescence from individual polymer particles. Immunoagglutination was initiated by mixing bovine serum albumin (BSA)-covered latex particles (of 1.8 mm in diameter) with anti-BSA IgG. The analysis of experimental data was performed on the basis of a mathematical model of diffusion-limited immunoagglutination aggregation with a steric factor. The steric factor was determined by the size and the number of binding sites on the surface of a latex particle. The obtained data are in good agreement with the proposed mathematical modeling. ---------- ---------- ---------- ----------