Peter Tarasov

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.

Light-scattering properties of individual erythrocytes

We have studied the light-scattering properties of human erythrocytes both experimentally and theoretically. 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 degrees. 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 degrees. 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.

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.

Measurement of Mammalian Erythrocyte Indices from Light Scattering with Scaning Flow Cytometer

The technique of scanning flow cytometery (SFC) was adopted for measurement of erythrocyte indices: volume, hemoglobin concentration and surface area. The method has been verified on two types of mammalian red cells: human and murine. In order to access distribution of values, precision and stability of inverse algorithm of reconstitution of size and refractive index of microsphere from light scattering angular dependency (indicatrix) have been increased performing analysis of Fourier spectrum of indicatrix. Volume - hemoglobin concentration (V-HbC) map was obtained following well-known procedure of isovolumetric sphering, presented by Technicon Instruments. Additionally new method for measurement of paired distribution of erythrocyte surface area (S) and hemoglobin content (Hb) was implemented via registration of spherical stage in the course of colloid-osmotic hemolysis. Approach to characterization with SFC of native red cells in nonspherical stage is demonstrated.

OPTICS OF ERYTHROCYTES

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

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.