Abstract
The knowledge of optical properties of biological cells is essential to interpret their interaction with light and to derive morphological information and parameters associated with cell function like the oxygen transport capacity of human red blood cells (RBCs). We present a method to determine the dependence between the refractive index (RI) of human RBCs and their intracellular hemoglobin (Hb) concentration from spectral extinction measurements of a cell suspension. The procedure is based on the analysis of the corresponding ensemble averaged extinction cross section {bar{{boldsymbol{C}}}}_{{bf{e}}{bf{x}}{bf{t}}}(lambda ). Thus far two complementary approaches have been taken to derive RIs of RBCs. The first one uses homogeneous macroscopic samples prepared by hemolysis for the destruction of the RBCs’ membranes and subsequent centrifugation. A second approach is the determination of RIs of single intact cells by microscopic investigation. These techniques are limited to a few discrete wavelengths or a rather narrow wavelength range. In addition most of these techniques require additional information about the concentration dependence. In contrast, our approach yields the RI increment with Hb concentration of intact, reversibly isovolumetrically sphered, oxygenated RBCs over a wide wavelength range from 290 nm to 1100 nm from macroscopic measurements.
Highlights
The complex refractive index (RI) of biological cells describes their interaction with light and depends on the concentrations and spatial distribution of a variety of intracellular molecules, correlated to the corresponding biological function
Homogeneous solutions of Hb can be obtained from red blood cells (RBCs) by breaking open the cell membranes and it is known that their complex RI depends on the Hb concentration cHb according to[2,27,28] n(λ, cHb) = nH2O(λ) + cHb[α(λ) + iγ(λ)], (1)
Human RBCs were isolated from fresh blood samples by washing as described in the “Materials and methods” section
Summary
The complex refractive index (RI) of biological cells describes their interaction with light and depends on the concentrations and spatial distribution of a variety of intracellular molecules, correlated to the corresponding biological function. RI measurements were presented for high-concentration bulk Hb solutions prepared from concentrated RBCs obtained from fresh blood samples which were hemolysed by freezing For these samples, Hb concentrations were in the 260 gL−1 to 306 gL−1 range[27,28,35]. In the 1950 s Barer and Joseph[1,2] compiled and reported values of α ≈ 0.19 mLg−1 for Hb solutions in the visible range, without resolving the wavelength dependence These values have been widely used in simulation and analysis of light scattering and microscopic data[14,22,36,37]. Recent studies employing microscopic techniques on Hb solutions in the visible[32] and single RBCs in the UV39 reported values of α ≈ 0.23 mLg−1
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