Abstract
Light scattering by single cells is widely applied for flow cytometric differentiation of cells. However, even for human red blood cells (RBCs), which can be modeled as homogeneous dielectric particles, the potential of light scattering is not yet fully exploited. We developed a dedicated flow cytometer to simultaneously observe the forward scattering cross section (FSC) of RBCs for orthogonal laser beams with incident wave vectors and . At a wavelength , bimodal distributions are observed in two-dimensional dot plots of FSC( ) vs. FSC( ), which result from the RBCs' random orientation around the direction of flow, as well as from the distributions of their size and their optical properties. Typically, signals of RBCs were analyzed. We actively oriented the cells in the cytometer to prove that orientation is the main cause of bimodality. In addition, we studied the wavelength dependence of FSC( ) using and 632.8 nm, covering both weak and strong light absorption by the RBCs. Simulations of the light scattering by single RBCs were performed using the discrete dipole approximation (DDA) for a range of sizes, orientations and optical properties to obtain FSC distributions from RBC ensembles. Using the axisymmetric biconcave equilibrium shape of native RBCs, the experimentally observed distributions cannot be reproduced. If, however, an elongated shape model is employed that accounts for the stretching of the cell by hydrodynamic forces in the cytometer, the features of the strongly bimodal measured frequency distributions are reproduced by the simulation. Elongation ratios significantly greater than 1 in the range of 1.5 to 2.5 yield the best agreement between experiments and simulated data.
Highlights
The shape and elastic properties of various body cells are known to change during disease
The latter indicates that the cells are oriented in a way, where they are asymmetric with respect to the direction of flow
Pronounced bimodal distributions were observed and are caused by the orientation of red blood cells (RBCs) in the flow channel of the cytometer and their elongated shape due to hydrodynamic forces. This interpretation was experimentally validated by actively orienting the erythrocytes using a steel capillary for the injection of RBCs in the sheath flow whose outlet was changed from a circular cross section to a flattened, oval cross section
Summary
The shape and elastic properties of various body cells are known to change during disease. Changes in cell deformability have been reported for various cancerous body tissues [1]. Red blood cells (RBCs) exhibit remarkable rheological characteristics that are important for their biological function of oxygen delivery to tissue and removal of carbon dioxide through the vascular system in a wide range of blood flow conditions [2]. Alterations of mechanical properties of human RBCs have been reported for peripheral vascular disease [3], sickle cell anemia [4], malaria [5,6], diabetes mellitus [7], sepsis and renal failure [8]. Other researchers reported in-flow measurements of light scattering patterns for the characterization of RBCs with data analysis based on rigorous wave-optical simulations [10,11,12]
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