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Project titleStudy of the morphological and functional properties of biological cells, including blood cells, using a sub-diffraction optical resolution and mathematical modeling of intra-and intercellular processes, in order to create new cell assays for clinical diagnosis

AffiliationVoevodsky Institute of Chemical Kinetics and Combustion Siberian Branch of the Russian Academy of Sciences,

Research area 05 - FUNDAMENTAL RESEARCH IN MEDICINE, 05-602 - Physical methods of medical diagnostics. Tomography

Keywordsflow cytometry, blood cells, light scattering, inverse problem, biokinetics, mathematical modelling

Annotation
Clinical diagnosis is the basis for disease prevention and possible treatment of patients. Fundamental knowledge can significantly enhance diagnostic capabilities and improve its quality. In addressing the problem of monitoring the functional state of the organism it is critical to select a nexus, which concentrates relevant information about the status of organs, cells and biomolecules. Blood cells are one of the main information-transfer agents between the body organs. These cells are classified into eight types, while the number of different biomolecules and organs is much larger. Any changes in organs or concentrations of biomolecules lead to changes in the blood cells characteristics. Thus, the most effective approach to monitoring is a comprehensive study of the morphological and functional properties of the eight types of blood cells. Modern analytical hematology offers only a rather limited set of characteristics: erythrocytes (concentration, volume , hemoglobin), platelets (concentration, volume), lymphocytes (concentration), monocytes (concentration), neutrophils (concentration), basophils (concentration) , eosinophils (concentration), and microparticles (-----). This set does not allow one to diagnose a number of fundamental changes in the body. Moreover, the precision of the existing analytical methods is also far from perfect. Thus, it is promising to study the cell morphology with sub-diffraction optical resolution to detect small deviations in the cell characteristics as they interact with each other and the surrounding biomolecules. The most effective approach to the analysis of biological cells is flow cytometry technology, which allows single-cell analysis in the flow cell at high speed. It is due to the high efficiency and reliability of such analysis that flow cytometry forms a basis for the vast majority of modern laboratory analyzers used for hematological, immunological, and immunochemical studies of blood in clinical laboratories. However, the current implementation of flow cytometers is far behind the modern capabilities of optics, electronics and mathematical processing of the experimental data. We propose to undertake a comprehensive study of biological cells with quantitative measurement of morphological and functional characteristics, using our latest fundamental achievements in optical analysis of single cells based on the original scanning flow cytometer, efficient theoretical method to simulate light scattering by cells of arbitrary shape and internal structure, and innovative approaches to solving inverse problems of light scattering and biokinetics. We plan to develop the mathematical model of native erythrocyte and reticulocyte shapes to improve stability of the solution of the inverse light-scattering problem for these cells. This will increase the number of determined morphological characteristics of erythrocytes (size, diameter, sphericity index, hemoglobin, membrane permeability, and elasticity) and improve the accuracy We also plan to continue studies of the nucleus structure influence on the light-scattering characteristics of mononuclear cells (lymphocytes, monocytes, stem cells). Using a new optical model for such cells should increase the amount of measured characteristics (cell and nucleus sizes, the optical density of the nucleus and cytoplasm, apoptotic index) and improve the accuracy of their determination. We plan to analyze the spatial distribution of the light scattered by the dimers of platelets to determine their characteristics (volume, sphericity index, optical density, activation and aggregation indices). As a result, we will justify the possibility of express identification and characterization of blood cells using an unique technology of scanning flow cytometry. http://cyto.kinetics.nsc.ru/

Expected results
1. Upgrade of the scanning flow cytometry to allow measurement of light scattering by cells at two wavelengths and extension of the measured light-scattering angular range into the back hemisphere (from 110 to 175 degrees). Currently, the Scanning Flow Cytometer (SFC) developed by project authors allows one to measure the light-scattering properties of single cells with world-record amount of information, namely in the range of scattering angles from 5 to 100 degrees and in different states of polarization (Strokotov et al. Cytometry Part A 2011; 79A: 570). We plan to modernize optical and electronic system of the SFC to increase the amount of light-scattering data collected from each cell, which in turn will increase the accuracy of both identification and characterization. Such upgrade will keep the authors at world-leading position in field of sub-diffraction (nanometer) optical characterization of cells. Based on the upgrade, we will prepare specifications for developing technical design of the production of a pilot batch of SFC. We also plan to publish two papers in international journals. 2. Study of the clustering and inter-nodal interpolation in combination with databases of light-scattering patterns to solve inverse light-scattering problems. Biological variability of the blood cell characteristics and the complexity of their optical models leads to a substantial increase in the required size of corresponding databases (Dyatlov et al Inverse Problems 2012; 28:045012; Gilev et al JQSRT 2013, 131:202-214). This slows down solution of the inverse problem. We plan to develop two algorithms to speed up the use of database and verify their performance in the analysis of biological particles. We also plan to publish two papers in international journals. 3. Study of light scattering properties of activated platelets and their dimers to identify those two classes during aggregation. Currently it is not possible to identify platelets dimers among the monomers due to large variability of their characteristics (volume, refractive index, shape). Moreover, platelet dimer formation takes place through the activation accompanied by the shape change, which also complicates the identification (Moskalensky et al. J. Biomed. Opt 2013; 18:017001). We plan to study light-scattering properties of platelet dimers using the upgraded SFC to measure the scattering intensity distribution in the backward hemisphere. We also plan to publish two papers in international journals. 4. The study of platelet activation to create and verify a mathematical molecular-kinetic model of platelet activation and their subsequent aggregation. Platelet activation and aggregation play a major role in such diseases as hemophilia and thrombophlebitis. Quantitative measurement of platelet activation is possible using the stimulated activation with registration of a fraction of activated cells. Created during this stage mathematical model of platelet activation will allow one to measure platelet activation and aggregation indices of patients. We also plan to publish two papers in international journals. 5. Kinetic studies of apoptosis of single cells by confocal microscopy and of their populations using scanning flow cytometry. There exists no quantitative description of apoptosis kinetics, based on the analysis of molecular and morphological measurements of cells. Qualitatively, cell apoptosis proceeds through the phase of chromatin condensation and reduction of the nucleus volume. Nucleus volume and the kinetics of its reduction can be measured by a SFC (Strokotov et al. J Biomed Opt 2009; 14:064036, Maltsev et al. Flow Cytometry: Principles, Methodology and Applications. NY: Nova Science Publishers; 2013, P 79-103). We plan to create molecular-kinetic model of apoptosis both for single cells and cell populations to quantify the susceptibility of the analyzed cells to apoptosis. We also plan to publish two papers in international journals. 6. Study of light-scattering properties of single native erythrocytes to improve the accuracy of determination of their characteristics (volume, membrane area, and hemoglobin concentration) and study of evolution of the characteristics distributions during erythrocyte lysis. There exists no universal mathematical description of the shape of a mature erythrocyte, which hampers the determination of these cells’ characteristics, for example, the degree of asphericity and membrane area, in clinical diagnostics. Study of light-scattering properties of erythrocytes will allow us to test the adequacy of several known shape models. We will then use the best model to solve the inverse light-scattering problem to determine the erythrocytes characteristics. Measuring the evolution of the distribution functions of these characteristics during lysis and using biokinetic models of this process we will determine the dynamic characteristics of the erythrocyte population, such as membrane ion permeability and cell elasticity. We also plan to publish two papers in international journals.

Annotation of the results obtained in 2016
Flow cytometry method (FCM) is widely used for analysis of cell-derived microparticles (MPs). Numerous efforts are currently aimed to standardize these measurements among different instruments. We push the FCM characterization of MPs to the limit based on rigorous simulation of measured signals. We measured forward- and side-scatter (FSC/SSC) signals and angle-resolved light-scattering profiles (LSPs) of polystyrene microspheres and MPs, including their aggregates, using a scanning flow cytometer (SFC). We used the Mie theory to (1) accurately evaluate instrument detection limits; (2) construct FSC/SSC gates for MPs in absolute scales of size and refractive index (RI); and (3) determine size and RI of individual spherical MPs. LSPs were used for advanced characterization, including differentiation of spherical and nonspherical particles. The proposed absolute FSC/SSC gating is naturally standardized for any FCM instrument, given the knowledge of its optical system and leads to instrument-independent analysis of MPs. The inverse Mie problem has a unique solution only for some regions of size and RI and uncertainties rapidly increase with decreasing size and RI. The developed methods are applicable to any flow cytometer, but are limited by assumption of particle sphericity. The latter can be relaxed only if additional signals, such as LSP, are measured. Whereas commercially available hematological analyzers measure volume of individual platelets, angle-resolved light-scattering provides unique ability to additionally measure their shape index. We utilized the scanning flow cytometer to measure light-scattering profiles (LSPs) of individual platelets taken from 16 healthy donors and the solution of the inverse light-scattering problem to retrieve the volume and shape index of each platelet. In normal conditions, the platelet shape index distribution (PSID) demonstrates three peaks, which relate to resting, partially activated, and fully activated platelets. We developed an algorithm, based on fitting PSID by a sum of three peak functions, to determine the percentage, mean platelet shape index, and distribution width of each platelet fraction. In total, this method gives eight additional parameters of platelet morphology and function to be used in clinical hematological analysis. We also stimulated the platelets with adenosine diphosphate (ADP) and measured the dependence of equilibrium PSID, including the total percentage of activated platelets, on ADP concentration. We propose a method for characterization of mature red blood cells (RBCs) morphology, based on measurement of light-scattering patterns (LSPs) of individual RBCs with the scanning flow cytometer and on solution of the inverse light-scattering (ILS) problem for each LSP. We considered a RBC shape model, corresponding to the minimal bending energy of the membrane with isotropic elasticity, and constructed an analytical approximation, which allows rapid simulation of the shape, given the diameter and minimal and maximal thicknesses. The ILS problem was solved by the nearest-neighbor interpolation using a preliminary calculated database of 605,000 theoretical LSPs. For each RBC in blood sample we determined three abovementioned shape characteristics and refractive index, which also allows us to calculate volume, surface area, sphericity index, spontaneous curvature, hemoglobin concentration and content.

Publications

1. Chernyshova E.S., Zaikina Y.S., Tsvetovskaya G.A., Strokotov D.I., Yurkin M.A., Serebrennikova E.S., Volkov L., Maltsev V.P., Chernyshev A.V. Influence of magnesium sulfate on HCO3/Cl transmembrane exchange rate in human erythrocytes Journal of Theoretical Biology, 393, 194–202 (year - 2016) https://doi.org/10.1016/j.jtbi.2015.12.023

2. Gilev K.V., Yurkin M.A., Chernyshova E.S., Strokotov D.I., Chernyshev A.V., and Maltsev V.P. Mature red blood cells: from optical model to inverse light-scattering problem Biomedical Optics Express, 7, 1305-1310 (year - 2016) https://doi.org/10.1364/BOE.7.001305

3. Konokhova A.I.; Chernova D.N.; Strokotov D.I.; Karpenko A.A.; Chernyshev A.V.;Maltsev V.P.; Yurkin M.A. Light-scattering gating and characterization of plasma microparticles Journal of Biomedical Optics, 21(11), 115003 (year - 2016) https://doi.org/10.1117/1.JBO.21.11.115003

4. Litvinenko A.L. Moskalensky A.E., Karmadonova N.A., Nekrasov V.M., Strokotov D.I., Konokhova A.I., Yurkin M.A., Pokushalov E.A., Chernyshev A.V., Maltsev V.P. Fluorescence‐free flow cytometry for measurement of shape index distribution of resting, partially activated, and fully activated platelets Cytometry Part A, 89: 1010–1016 (year - 2016) https://doi.org/10.1002/cyto.a.23003

5. Polshchitsin A.A., Nekrasov V.M., Zakovryashin V.S., Yakovleva G.E., Maltsev V.P., Yurkin M.A., Chernyshev A.V. Kinetic turbidimetry of patchy colloids aggregation: latex particles immunoagglutination Colloids and Surfaces A: Physicochemical and Engineering Aspects, - (year - 2016) https://doi.org/10.1016/j.colsurfa.2016.12.018

6. Yurkin M.A., Huntemann M. Rigorous and Fast Discrete Dipole Approximation for Particles near a Plane Interface The Journal of Physical Chemistry C, 119, 29088–29094 (year - 2015) https://doi.org/10.1021/acs.jpcc.5b09271

7. Mishchenko M.I., Dlugach J.M., Yurkin M.A., Bi L., Cairns B., Liu L., Panetta R.L., Travis L.D., Yang P., and Zakharova N.T. First-principles modeling of electromagnetic scattering by discrete and discretely heterogeneous random media Physics Reports, 632: 1–75 (year - 2016) https://doi.org/10.1016/j.physrep.2016.04.002

8. - Диагноз ясен Наука в Сибири, - (year - )

9. - Этот аппарат спасёт тысячи жизней! Что изобрели новосибирские учёные? 8 Канал.Новосибирск, - (year - )

10. - Кровавая история Наука в Сибири, - (year - )

11. - Компьютерная программа новосибирских ученых поможет распознать «сигнал болезни» Вести-Новосибирск, - (year - )

Annotation of the results obtained in 2014
We present a method for the quantitative characterization of an early-phase nuclear apoptotic volume decrease (AVD) in chemically-induced apoptosis in single live-cells observed by 3D time-lapse confocal microscopy. The method is based on a newly developed algorithm to process microscopy data set and mathematical model formulated on the basis of a set of kinetic equations which describe a dynamic of nuclear AVD driven by peripheral chromatin condensation. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shown to produce accurate results in comparison with more sophisticated approaches.

Publications

1. - Enhanced characterization of milk fat globules by their size, shape and refractive index with scanning flow cytometry International Dairy Journal, Volume 39, Issue 2, December 2014, Pages 316–323 (year - 2014) https://doi.org/10.1016/j.idairyj.2014.08.006

2. - Additivity of light-scattering patterns of aggregated biological particles Journal of Biomedical Optics, V. 19, 085004 (year - 2014) https://doi.org/10.1117/1.JBO.19.8.085004

3. - Brownian aggregation rate of colloid particles with several active sites The Journal of Chemical Physics, V. 141, 064309 (year - 2014) https://doi.org/10.1063/1.4892163

4. - Size-dependent optical properties of polyethylene powders in far-IR region: On the way to universal matrix Journal of Quantitative Spectroscopy and Radiative Transfer, Volume 147, November 2014, Pages 1–7 (year - 2014) https://doi.org/10.1016/j.jqsrt.2014.05.011

Annotation of the results obtained in 2015
Flow cytometry is a powerful technique for analysis of individual blood cells and microparticles. However, only rough estimation of sizes has been possible for the latter based on standard measurement of forward and side scattering. We showed that measurement of a full scattering profile for each microparticle with an advanced set-up, a scanning flow cytometer, leads to determination of its shape, refractive index and size with nanometer precision. This not only allowed unambiguous separation of larger microparticles from blood platelets, but also clearly identified classes of microparticles having different refractive indices and shapes. It is unprecedented precision of the latter that opens a way for objective classification and characterization of microparticles of various origin and/or composition. Distribution of platelets over the shape aspect ratio revealed two distinct populations of resting (flattened) and activated (rounded) cells. The fraction of rounded cells increased with the increasing agonist concentration. Dynamics of platelet shape change closely correlated with the intracellular calcium concentration. We propose a simple model of shape change based on the excessive curvature of the marginal band of microtubules, due to the increase of intracellular calcium, which bends the band into a 3-dimensional rounded structure called the “overcurved ring”. The results support the use of platelet aspect ratio as a flow-cytometric parameter to be applied for the clinical evaluation of platelet function and for investigation of the mechanics of platelet shape change. The proposed mechanical model provides novel knowledge about platelets and may help in the development of therapeutic strategies aimed at modification of platelet function. Magnesium sulfate (MgSO4) is widely used in medicine but molecular mechanisms of its protection through influence on erythrocytes are not fully understood and are considerably controversial. Using scanning flow cytometry, in this work for the first time we observed experimentally (both in situ and in vitro) a significant increase of HCO3-/Cl- transmembrane exchange rate of human erythrocytes in the presence of MgSO4 in blood. For a quantitative analysis of the obtained experimental data, we introduced and verified a molecular kinetic model, which describes activation of major anion exchanger Band 3 (or AE1) by its complexation with free intracellular Mg2+ (taking into account Mg2+ membrane transport and intracellular buffering). Fitting the model to our in vitro experimental data, we observed a good correspondence between theoretical and experimental kinetic curves that allowed us to evaluate the model parameters and to estimate for the first time the association constant of Mg2+ with Band 3 as KB~0.07 mM, which is in agreement with known values of the apparent Mg2+ dissociation constant (from 0.01 to 0.1 mM) that reflects experiments on enrichment of Mg2+ at the inner erythrocyte membrane (Gunther, 2007). Results of this work partly clarify the molecular mechanisms of MgSO4 action in human erythrocytes. The method developed allows one to estimate quantitatively a perspective of MgSO4 treatment for a patient. It should be particularly helpful in prenatal medicine for early detection of pathologies associated with the risk of fetal hypoxia.

Publications

1. - Dynamic quantification of antigen molecules with flow cytometry Journal of Immunological Methods, 418, 66–74 (year - 2015) https://doi.org/10.1016/j.jim.2015.02.001

2. - Rectangular dipoles in the discrete dipole approximation Journal of Quantitative Spectroscopy& Radiative Transfer, v. 156, pp. 157-169 (year - 2015) https://doi.org/10.1016/j.jqsrt.2015.01.019

3. - Time-domain discrete-dipole approximation for simulation of temporal response of plasmonic nanoparticles OPTICS EXPRESS, Vol. 23, No. 12, p. 15555 (year - 2015) https://doi.org/10.1364/OE.23.015555

4. Konokhova A.I., Chernova D.N., Moskalensky A.E., Strokotov D.I., Yurkin M.A., Chernyshev A.V., Maltsev V.P. Super-Resolved Calibration-Free Flow Cytometric Characterization of Platelets and Cell-Derived Microparticles in Platelet-Rich Plasma Cytometry Part A, Vol. 89, № 2. P. 159–168. (year - 2016) https://doi.org/10.1002/cyto.a.22621