Konstantin V. Gilev

Is there a difference between T- and B-lymphocyte morphology?

We characterize T- and B-lymphocytes from several donors, determining cell diameter, ratio of nucleus to cell diameter, and refractive index of the nucleus and cytoplasm for each individual cell. We measure light-scattering profiles with a scanning flow cytometer and invert the signals using a coated sphere as an optical model of the cell and by relying on a global optimization technique. The main difference in morphology of T- and B-lymphocytes is found to be the larger mean diameters of the latter. However, the difference is smaller than the natural biological variability of a single cell. We propose nuclear inhomogeneity as a possible reason for the deviation of measured light-scattering profiles from real lymphocytes from those obtained from the coated sphere model.

Comparison of the discrete dipole approximation and the discrete source method for simulation of light scattering by red blood cells.

The discrete sources method (DSM) and the discrete dipole approximation (DDA) were compared for simulation of light scattering by a red blood cell (RBC) model. We considered RBCs with diameters up to 8 mum (size parameter up to 38), relative refractive indices 1.03 and 1.06, and two different orientations. The agreement in the angle-resolved S(11) element of the Mueller matrix obtained by these methods is generally good, but it deteriorates with increasing scattering angle, diameter and refractive index of a RBC. Based on the DDA simulations with very fine discretization (up to 93 dipoles per wavelength) for a single RBC, we attributed most of the disagreement to the DSM, which results contain high-frequency ripples. For a single orientation of a RBC the DDA is comparable to or faster than the DSM. However, the relation is reversed when a set of particle orientations need to be simulated at once. Moreover, the DSM requires about an order of magnitude less computer memory. At present, application of the DSM for massive calculation of light scattering patterns of RBCs is hampered by its limitations in size parameter of a RBC due to the high number of harmonics used for calculations.

Mature red blood cells: from optical model to inverse light-scattering problem

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 250,000 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.

An optimization method with precomputed starting points for solving the inverse Mie problem

We study the inverse light-scattering problem which arises in the characterization of small particles by means of scanning flow cytometry. The problem is stated in general form as the problem of solution of a nonlinear equation and solved by the gradient optimization method. In this event, the problem of choosing the starting point appears. In this paper, we propose a method for making this choice based on the preliminary analysis of the direct map. A number of numerical examples are given, using both synthetic and experimental data.

The scanning flow cytometer modified for measurement of two-dimensional light-scattering pattern of individual particles

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.

Advanced consumable‐free morphological analysis of intact red blood cells by a compact scanning flow cytometer

Whereas modern automated blood cell analyzers measure the volume of individual red blood cells (RBCs), leading to four RBC indices (mean corpuscular volume, MCV; mean corpuscular hemoglobin, MCH; mean corpuscular hemoglobin concentration, MCHC; red cell distribution width, and RDW), the RBC shape has not been assessed by clinical screening tools. We applied the scanning flow cytometer (SFC) for complete characterization of intact RBC morphology in terms of diameter, maximal and minimal thicknesses, volume, surface area, sphericity index, spontaneous curvature, hemoglobin concentration, and content. The above‐mentioned individual RBC characteristics were measured without fluorescent markers and other chemicals by a SFC equipped only with 660 nm laser for RBC illumination and single detector for measurement of angle‐resolved light scattering. The distributions over all RBC characteristics were constructed and processed statistically to form the novel 31 RBC indices for 22 donor samples. Our results confirm the possibility of precise, label‐free, enhanced morphological analysis of individual intact RBCs with compact single‐detector flow cytometer. Detailed characterization of RBCs with high statistics and precision can be used to increase the value of screening examinations and to reveal pathologies accompanied by abnormality of RBC shape.

Optimization methods for characterization of single particles from light scattering patterns

We address the inverse light-scattering problem for particles described by a several-parameters model, when experimental data are given as an angle-resolved light- scattering pattern (LSP). This problem is reformulated as an optimization (nonlinear re- gression) problem, for which two solution methods are proposed. The first one is based on standard gradient optimization method, but with careful choice of the starting point. The second method is based on precalculated database of theoretical LSPs, from which the closest one to an experimental LSP is selected for characterization. We tested both methods for characterization of polystyrene microspheres using a scanning flow cytometer (SFC).