Flickering Analysis of Erythrocyte Mechanical Properties: Dependence on Oxygenation Level, Cell Shape, and Hydration Level

Abstract

Erythrocytes (red blood cells) play an essential role in the respiratory functions of vertebrates, carrying oxygen from lungs to tissues and CO(2) from tissues to lungs. They are mechanically very soft, enabling circulation through small capillaries. The small thermally induced displacements of the membrane provide an important tool in the investigation of the mechanics of the cell membrane. However, despite numerous studies, uncertainties in the interpretation of the data, and in the values derived for the main parameters of cell mechanics, have rendered past conclusions from the fluctuation approach somewhat controversial. Here we revisit the experimental method and theoretical analysis of fluctuations, to adapt them to the case of cell contour fluctuations, which are readily observable experimentally. This enables direct measurements of membrane tension, of bending modulus, and of the viscosity of the cell cytoplasm. Of the various factors that influence the mechanical properties of the cell, we focus here on: 1), the level of oxygenation, as monitored by Raman spectrometry; 2), cell shape; and 3), the concentration of hemoglobin. The results show that, contrary to previous reports, there is no significant difference in cell tension and bending modulus between oxygenated and deoxygenated states, in line with the softness requirement for optimal circulatory flow in both states. On the other hand, tension and bending moduli of discocyte- and spherocyte-shaped cells differ markedly, in both the oxygenated and deoxygenated states. The tension in spherocytes is much higher, consistent with recent theoretical models that describe the transitions between red blood cell shapes as a function of membrane tension. Cell cytoplasmic viscosity is strongly influenced by the hydration state. The implications of these results to circulatory flow dynamics in physiological and pathological conditions are discussed.

Measurement Techniques for Red Blood Cell Deformability: Recent Advances

“…The bending modulus B of RBCs does not significantly change with temperature (Nash and Meiselman, 1985) or cell Hb concentration for both normal and sickle cells (Evans, Mohandas et al, 1984). The recent experiments (Betz, Lenz et al, 2009;Yoon, Hong et al, 2009) have also measured that the bending modulus of RBCs is of the order of 50 kbT. However, several other techniques have measured lower bending moduli of RBC membranes.…”

“…Cytosolic viscosity depends on the concentration and viscosity of Hb. By measuring the dynamic contour fluctuations of RBC membrane, the cytoplasmic viscosity has been obtained in the range of 2-5 mPa·s (Yoon, Hong et al,2009). Recently, the dynamic membrane fluctuation measurements retrieved the cytoplasmic visocity of the RBCs at physiological osmotic pressure as 5-6 mPa·s, and the cytosol viscosity increases monotonically from with increasing osmolality .…”

Equilibrium physics breakdown reveals the active nature of red blood cell flickering

“…Today, flickering analysis is envisaged as a promising tool for high-rate malaria diagnosis 4 . However, although equilibrium fluctuation models [5][6][7] have been used to infer mechanical properties of RBCs since the seminal work of Brochard & Lennon 8 , the fundamental question whether membrane fluctuations are driven by an active process or simply by thermal agitation remains controversial [9][10][11][12] .…”

Plasmodium falciparum erythrocyte-binding antigen 175 triggers a biophysical change in the red blood cell that facilitates invasion

“…To determine the cause of biophysical changes seen by RT-DC, we sought to use a method that would permit independent measurement of the tension and bending modulus of the RBC surface on EBA175 binding. Membrane fluctuation analysis (flicker spectroscopy) has been used extensively as a method for mechanical characterization of vesicles (25,26) and erythrocytes (27)(28)(29) at the single-cell level. Critically for our purposes, the cytoskeletal and lipid membrane properties dominate different ranges of the fluctuation spectrum.…”

“…S1). Conversely, variations in the RBC bending modulus are largely expected to come from changes to the cell membrane, and this parameter is reflected in the higher mode numbers (27) (Fig. S1).…”