Polarized Raman spectroscopic investigations on hemoglobin ordering in red blood cells

Abstract

We have investigated the dependence of the Raman spectrum of an optically trapped red blood cell (RBC) on the orientation of the cell, relative to the polarization direction of the Raman excitation beam. The Raman scattered light polarized parallel to the polarization direction of the excitation beam was observed to depend upon the orientation of the cell. In particular, the heme bands at ∼754 cm⁻¹ and in the 1500 to 1700 cm⁻¹ region were observed to become maximum when the cells' equatorial plane was parallel to the excitation beam polarization direction and minimum when the cells' plane was normal to the polarization direction. In contrast, no significant orientational dependence was seen in the Raman scattered light polarized orthogonal to the polarization direction of the excitation beam. Theoretical simulations carried out to investigate these observations suggest that inside the RBCs, the hemoglobin molecules must be present in an ordered arrangement, such that heme-porphyrin planes become preferentially orientated parallel to the RBCs' equatorial plane.

Polarisation optics for biomedical and clinical applications: a review

“…Here we briefly summarise common phantoms used for biomedical polarimetric techniques. These techniques include polarised wide-field microscopy 16 , 24 , 183 , polarised light spatial frequency imaging 184 , polarimetric endoscopy 185 – 190 , spectral light scattering polarimetry 18 , 82 , 191 – 193 , polarised fluorescence spectroscopy 194 – 196 , polarised confocal microscopy 197 , polarised Raman-spectroscopy 198 , 199 , polarised super-resolution microscopy 154 , 155 , polarisation sensitive optical coherence tomography 200 – 218 , non-diffraction beam polarimetry (such as Bessel beam based) 219 , polarisation-resolved nonlinear microscopy (including second/third harmonic generation) 220 – 226 , and polarised speckle imaging 213 , 227 (several techniques will be mentioned again in the Discussion). The relationship between incoherence and depolarisation of the light should be kept in mind when considering coherence based polarimetric techniques: they are different but related optical concepts.…”

Raman Spectroscopy of Blood and Blood Components

“…The results of theoretical simulations suggested that hemoglobin ''must be present in an ordered arrangement, such that the hemeporphyrin planes are preferentially orientated parallel to the RBCs' equatorial plane''. 59 Laser tweezers Raman spectroscopy has become an increasingly important technique in the study of living RBCs, for investigations of oxygenation within single cells, [60][61][62] and for investigation of the effects of laser light on RBC biology. [63][64][65] The method has also been integral for characterizing red cell response to stimulus/ stress (e.g., alcohol-induced denaturation, 66 electrical current, 67 oxidative stress, 68 and pH changes 69 ), and has been deployed to compare oxygen uptake of different globin-containing cells.…”

Optical Properties of Glycated and Non-Glycated Hemoglobin – Raman/Fluorescence Spectroscopy and Refractometry

“…Based on theoretical results in Ref. [39] hemoglobin exists in an ordered arrangement, such that the heme-porphyrin planes are preferentially orientated parallel to the RBCs' equatorial plane. The glycation of red blood cells can be precisely estimated using the electric/dielectric losses and polarization changes, described in detail in Ref.…”