Raman excitation wavelength investigation of single red blood cells in vivo

Abstract

Raman spectra are reported for oxygenated and deoxygenated haemoglobin contained within a single red blood cell in vivo using excitation wavelengths of 488, 514, 568 and 632.8 nm. The peak assigned in previous work to n 4 is observed at 1376 cm −1 in oxygenated cells and 1356 cm −1 in deoxygenated cells with the results from 488 nm excitation consistent with earlier Raman studies on isolated haem proteins. Exciting the cells with 514 nm radiation revealed two bands appearing in this region at 1372 and 1356 cm −1 in the oxygenated state, whereas in the deoxygenated state only one band at 1356 cm −1 is observed. At 632.8 nm excitation bands in the n 4 region appeared at 1367 and 1365 cm −1 in the oxygenated and deoxygenated states, respectively. Our results clearly show that the enhancement of bands in the vicinity of n 4 within single erythrocytes is influenced by the excitation wavelength. Furthermore, many other bands observed in oxygenated erythrocytes using 632.8 nm excitation were dramatically enhanced compared with the bands observed with other excitation wavelengths. Ruling out other explanations, it is hypothesized that the enhancement observed at 632.8 nm results from excitonic coupling between aligned porphyrins. The high concentration of haemoglobin in a single cell enables the porphyrins to be in close proximity to permit charge transfer between the haem moieties. The high signal-to-noise ratio and excellent reproducibility obtained using Raman water immersion microspectroscopy on single erythrocytes in vivo shows potential as a diagnostic tool for a variety of haemopathies. However, judicious choice of the excitation wavelength is a prerequisite especially if the technique is applied to diagnose oxidation status within erythrocytes.

Probing differentiation in cancer cell lines by single-cell micro-Raman spectroscopy

“…The observed vibrational frequencies and respective assignments are summarized in Table 1; these assignments are based on previously reported values of vibrational frequencies in biological molecules. [29][30][31][32][33][34][35][36][37][38][39][40] Figure 5 shows the difference in marker peak intensities for undifferentiated and differentiated cells with standard deviation to indicate the data spread in our experiments. It is clear from Figs.…”

Raman Spectroscopy of Blood and Blood Components

“…Brémard et al used RRS to characterize hemozoin 92 and Ong et al used RRS to characterize infected RBCs. 93,94 It was mentioned above (Intracellular hemoglobin) that workers at Monash University carried out some of the earliest studies of intracellular hemoglobin 16,39,41 and that, in the years since, two members of the team, Wood and McNaughton, have gone on to do much Raman spectroscopic analysis of pathological erythrocytes. In 2003, Wood, McNaughton, and co-workers reported an enhancement of a particular hemozoin Raman band (at 1374 cm…”

“…13 and assignments are based on a porphyrin labeling scheme created by Abe et al 14 Data is collected from studies by Wood et al 15 and Wood and McNaughton. 16 Positions vary slightly with excitation wavelength, and some modes are specific to oxygenation state. Intensities of bands depend on resonance enhancement mechanisms.…”

Confocal Raman microscopy on single living young and old erythrocytes

“…The line mapping was performed using the 1358 cm 21 (m4) band for Hb under the 514.5-nm excitation light, for its signal is strong and almost without variation under the measuring condition, 10 from the m4 band one can also obtain the information about the electron population in the p* orbital. 1,8,21 In line mapping, the scan step was set to 0.5 lm, the laser power at sample was 0.6 mW, and the other scan parameters were the same as mentioned above.…”

“…Hemoglobin is very sensitive to Raman scattering and the high symmetry and chromophoric structure of its heme result in strong Raman scattering and a rich spectrum for different laser excitation wavelengths. The hemoglobin's Raman characteristics in fully oxygenated (R) and deoxygenated (T) states, and in the circumstances of heme aggregation and denaturation, as well as the Raman imaging of functional erythrocytes [8][9][10][11][12][13] have been studied. Some interesting results were observed and the validity of Raman microspectroscopy on the cell was also proved.…”