Spectral Markers of Erythrocytes on Solid Substrate

Abstract

Proposed in previous paper [1,2] the new nondestructive method of optical microscopy allows to examine the structures of living cells (human erythrocytes) in their natural colors without its staining by using a specially designed substrate for deposition of biological sample and observing a native blood smears in reflected light. Color interference contrast image is achieved due to special condition of experiment is connected with chose of angle of incidental light, wave length of light of reflected ray, chemical composition of sample, thickness of sample, refractive index of sample, refractive index of substrate, chemical composition of substrate [1,2]. We can identify chemical compounds of erythrocytes after calibration color scale by alternative methods. For comparison we used Synchrotron Radiation based Fourier Transformed Infrared (SR-FTIR) microspectroscopy. By focusing of infrared beam of FTIR microscope on cell surface we can screen and distinguish difference erythrocytes by its color. For example on Fig. 49.1 we can see two neighbored erythrocytes where one of them have red color (point 1) and other-green (point 5). To identify their spectral markers we measured IR absorption spectra of cells at different points (1,2,3,4 and 5). Intermediated area (points 3 and 4) correspond to substrate spectra (silicon substrate) and their spectra are same. The peaks at 2,850 and 2,920 cm⁻¹ correspond mainly to the CH₂ stretching modes of the methylene chains in membrane lipids. At 1,650 cm⁻¹ the amide I band is observed, which results, principally, from the n(CO) stretching vibrations of the protein amide bonds; the amide II band, near 1,550 cm⁻¹, is a combination of the d(N–H) bending and n(C–N) stretching vibrations of the amide bonds. The peaks at 2,850 and 2,920 cm⁻¹ correspond mainly to the CH₂ stretching modes of the methylene chains in membrane lipids [3. The intensities of the absorption bands at 2,920 and 2,850 cm⁻¹ in green erythrocyte (point 5) were also significantly increased compared to red one (point 1). The protein conformational change of the red and green cells could be expressed as the ratio of the absorbance intensity at wavenumber 1650/1550, which represented the intensity of the amide II band of cells. Lipids have long hydrocarbon chains, so the dominant features in the IR spectrum of lipids are attributed to the asymmetric and symmetric stretching vibrations of CH₂ (2,920 and 2,850 cm⁻¹) groups in the molecule. Other biological components, such as proteins, nucleic acids, and carbohydrates, also have unique IR spectra.