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-O(2) 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 a(iu)(pi)-->d(xz)+O(2)(pi(g)) 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.

Raman spectroscopy: techniques and applications in the life sciences

“…Cytochrome c is a hemeprotein found in mitochondria, and its dynamics have been studied in apoptosing cells using resonance Raman of the 750 cm −1 pyrrole ring breathing band [113]. The oxygenation state of hemoglobin in single red blood cells can be also be observed with 785 nm-excited resonance Raman, in particular the Fe-O 2 stretching mode at 570 cm −1 was only seen for oxygenated cells [114].…”

Novel chiroptical analysis of hemoglobin by surface enhanced resonance Raman optical activity spectroscopy

“…It is well known that vibrations of pyrrol bonds, especially pyrrol half-ring vibrations, are very sensitive to the redox status of the heme Fe ion, and to the presence of the sixth ligand (O 2 , NO, CO). 24,29,30,35 Hence, it is possible that any change in Hb saturation with O 2 will be manifested in changes in the intensity or the position of the m 4 peak. However, specific designed experiments are needed to verify the sensitivity of SERROA signal (over SERRS) to specific changes in Hb conformation and oxygenation.…”

“…[24][25][26][27] Studying the Hb properties inside a living erythrocyte under normal pathological conditions has also been shown possible by using the resonance RS (RRS). [28][29][30] Isolated Hb has also been studied by SERS, 8,31 and in both reports the Ag colloids were prepared by reduction of Nacitrate, 32 and they employed 514 nm excitation laser. This excitation lies in the shorter wavelength of the Hb absorption, yielding preresonance Raman, while longer wavelength, e.g.…”

A self-filling microfluidic device for noninvasive and time-resolved single red blood cell experiments

“…[14][15][16] As an alternative to standard optical methods, Raman microscopy has emerged as a powerful technique to investigate not only the mechanical properties but also the molecular processes within single RBCs. [17][18][19][20][21] So-called Raman tweezers, which is the coupling of Raman spectroscopy with optical tweezers, have been successfully used to distinguish between healthy and diseased cells in thalassemia. 21,22 All approaches to single-cell experiments are based on the same principle, the combination of a method to separate and isolate individual cells from the bulk with a technique to extract the relevant biological or biophysical information.…”