High‐resolution Raman imaging reveals spatial location of heme oxidation sites in single red blood cells of dried smears

Abstract

Raman confocal microscopy with 488 nm excitation wavelength supported with atomic force microscopy (AFM), scanning near‐field optical microscopy (SNOM) and UV–Vis spectrometry was used to investigate air‐dried erythrocytes (red blood cells, RBCs) in whole human blood smears. The central internal part of the cell was dominated by the laser‐induced O2 dissociated oxyHb form as evidenced by the Fe2+ marker band appearing at 1356 cm−1. The existence of a thin outer layer of hemoglobin in the periphery of RBCs was assigned to hemichrome. Evidence for hemichrome includes the oxidation state marker band appearing at 1376 cm−1, the absence of FeO2 band at 570 cm−1 and a UV–Vis spectrum consistent with hemichrome. This is the first time that distributions of Fe2+/Fe3+ hemes inside the single RBC have been reported. The outer layer formation of hemichrome was additionally studied when RBCs were in contact with leucocytes and carotenoids of blood plasma. Copyright © 2014 John Wiley & Sons, Ltd.