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.

Visible Raman excitation laser induced power and exposure dependent effects in red blood cells

“…Since these changes were found to be irreversible (Figure B) and also accompanied by distortions in the cell morphology (Figure B) therefore suggest the formation of some photodamaged form of Hb e.g. hemichrome which has spectrum very similar to the oxy Hb . At higher power levels e.g.…”

The Role of Free Radicals in Hemolytic Toxicity Induced by Atmospheric‐Pressure Plasma Jet

“…Different excitation wavelengths bring differential Raman spectra of RBCs, due to the specific absorption bands of RBCs [ 23 ]. In previous reports, the main Raman spectral bands of RBCs were mostly assigned to hemoglobin vibration energy, and some were to cell membrane lipids and proteins [ 23 , 24 ]. Figure 4 shows the Raman spectra of RBCs after 5 minutes of APPJ treatment.…”

“…In the case of 488 nm laser ( Figure 4(a) ), the peak at 1110 cm −1 became lower (corresponding to acyl chain stretch↓), the peak at 1370 cm −1 became sharper and higher (corresponding to pyrrol ring vibration: 1355 cm −1 representing Fe 2+ ↓ and 1375 cm −1 representing Fe 3+ ↑), and the wide band of multiple peaks at 1550–1650 cm −1 (1530 cm −1 for C = C Trp↓, 1545 cm −1 for Fe 2+ ↓, 1608 cm −1 for Fe 2+ ↓, and 1642 cm −1 for Fe 3+ ↑) were modified by air APPJ treatment [ 24 ]. The peak at 2712 cm −1 was an overtone of 1355 cm −1 , whose intensities reduced together.…”

Recent advances in linear and non‐linear Raman spectroscopy. Part X

“…This is the first time that distributions of Fe 2+ /Fe 3+ hemes inside a single RBC have been reported. The outer layer formation of hemichrome was additionally studied when RBCs were in contact with leukocytes and carotenoids of blood plasma …”