Methods to investigate the erythrocyte membrane biophysical properties relevant to blood circulation

Erythrocytes (red blood cells) are critical for the transport of oxygen and carbon dioxide around the body, reaching all tissues ultimately through narrow capillaries which requires significant deformations of the cells. As a result, they must withstand continuous mechanical stress and are specifically adapted with a lipid bilayer enriched in cholesterol and an underlying spectrin-based cytoskeleton for stability and flexibility. Thus, erythrocyte membranes have unique mechanical, biophysical and electrostatic properties suited to their function, which have long been of physiological and biomedical interest. These include membrane elasticity and deformability (bending rigidity or elasticity) as well as membrane fluidity and tension, which depend on the lipid and protein composition of the membrane. This review details the methods available to measure these properties. Measurement of lipid bilayer fluidity or polarity commonly uses solvatochromic dyes (e.g. laurdan), while dyes responsive to membrane tension include Flipper-TR, a fluorescence lifetime imaging (FLIM) reporter. Transmembrane potential and membrane dipole potential can be measured by partitioning of lipophilic dyes and ratiometric fluorescence imaging, respectively. Analysis of spatial fluctuations, driven by thermal energy, can be monitored non-invasively by advanced imaging techniques and provide information on membrane elasatic parameters. A variety of mechanical approaches that cause perturbation of the membrane and measure its resistance have also been used, including micropipette aspiration, optical tweezers, atomic force microscopy (AFM). The principles, advantages and challenges of these methods for understanding erythrocyte membrane are discussed, as well as cell stresses and diseases that affect them.