Resonance Raman spectroscopy of red blood cells using near-infrared laser excitation

Abstract

Resonance Raman spectra of oxygenated and deoxygenated functional erythrocytes recorded using 785 nm laser excitation are presented. The high-quality spectra show a mixture of enhanced A_1g, A_2g, B_1g, B_2g, E_u and vinyl modes. The high sensitivity of the Raman system enabled spectra from four oxygenation and deoxygenation cycles to be recorded with only 18 mW of power at the sample over a 60-minute period. This low power prevented photo-/thermal degradation and negated protein denaturation leading to heme aggregation. The large database consisting of 210 spectra from the four cycles was analyzed with principal components analysis (PCA). The PC1 loadings plot provided exquisite detail on bands associated with the oxygenated and deoxygenated states. The enhancement of a band at 567 cm⁻¹, observed in the spectra of oxygenated cells and the corresponding PC1 loadings plot, was assigned to the Fe–O₂ stretching mode, while a band appearing at 419 cm⁻¹ was assigned to the Fe–O–O bending mode based on previous studies. For deoxygenated cells, the enhancement of B_1g modes at 785 nm excitation is consistent with vibronic coupling between band III and the Soret transition. In the case of oxygenated cells, the enhancement of iron-axial out-of-plane modes and non-totally symmetric modes is consistent with enhancement into the y,z-polarized transition ${\text{a}}_{{{\text{iu}}}} {\left( {\text{ $ \pi $ }} \right)} \to {\text{d}}_{{{\text{xz}}}} + {\text{O}}_{{\text{2}}} {\left( {{\text{ $ \pi $ }}_{{\text{g}}} } \right)}$ centered at 785 nm. The enhancement of non-totally symmetric B_1g modes in oxygenated cells suggests vibronic coupling between band IV and the Soret band. This study provides new insights into the vibrational dynamics, electronic structure and resonant enhancement of heme moieties within functional erythrocytes at near-IR excitation wavelengths.