Konstantin A. Semyanov

Experimental and theoretical study of light scattering by individual mature red blood cells by use of scanning flow cytometry and a discrete dipole approximation

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.

Single-particle sizing from light scattering by spectral decomposition

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 spectrums 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 microm at a wavelength of 0.633 microm). 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 microm and a sample of isovolumetrically sphered erythrocytes with a scanning flow cytometer to compare the accuracy of our new method with that of others.

Determination of volume, shape and refractive index of individual blood platelets

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.

Characterisation of Bio-Particles from Light Scattering

The primary aim of this monograph is to provide a systematic state-of-the-art summary of the light scattering of bioparticles, including a brief consideration of analytical and numerical methods for computing electromagnetic scattering by single particles, a detailed discussion of the instrumental approach used in measurement of light scattering, an analysis of the methods used in solution of the inverse light scattering problem, and an introduction of the results dealing with practical analysis of biosamples. Considering the widespread need for this information in optics, remote sensing, engineering, medicine, and biology, the book is useful to many graduate students, scientists, and engineers working on various aspects of electromagnetic scattering and its applications.

Discrimination of granulocyte subtypes from light scattering: theoretical analysis using a granulated sphere model

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.

OPTICS OF ERYTHROCYTES

We present optical methods to study erythrocytes and consider selected models to compute their light scattering.

Absolute real-time determination of size and refractive index of individual microspheres

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 m to m 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 X refractive index) was created for polystyrene latex and milk fat particles.

OPTICS OF LEUCOCYTES

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.

Scanning flow cytometer modified to distinguish phytoplankton cells from their effective size, effective refractive index, depolarization, and fluorescence

A laser flow cytometer based on scanning flow cytometry has been assembled. The unpolarized and linearly polarized light-scattering profiles, as well as the side emitted light in different spectral bands, were measured, allowing the simultaneous and real-time determination of the effective size and the effective refractive index of each spherelike particle. Additionally, each particle could be identified from depolarization and fluorescence measured simultaneously. The tests with aqueous samples of polystyrene spheres, fluorescent or nonfluorescent, and phytoplankton cells demonstrate that the system is able to retrieve size and refractive index with an accuracy of 1% and that the depolarization and fluorescence measurements allow the classification of particles otherwise indistinguishable.

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.