Robust High-Throughput Flickering Spectroscopy for Measurements of Red Blood Cell Membrane Mechanics

The mechanical properties of red blood cell (RBC) membranes are critical to their function in oxygen delivery, and changes to these properties as RBCs age affect their journey around the circulatory system, including clearance by the spleen. Such changes can also have significant health effects,including in cardiovascular disease and on the interactions between RBCs and malaria parasites. The function of RBCs requires them to be very soft, which together with the small size of the cells brings the energy required for measurable deformation of the membrane into the range of typical thermal energies. Red Blood Cells can be observed to flicker under optical microscopy, and these shape fluctuations can be quantified and used to obtain key biophysical parameters such as the tension and bending modulus of the membrane. Typically the shape of the cell's equator is extracted, and the mean power spectrum is obtained by time-averaging the power present in the normal modes of the thermal fluctuations. Flickering spectroscopy has been used extensively on RBCs, but has so far been very low throughput and with some technical limitations. Here we address issues related to active versus passive fluctuations, focusing and optics, camera exposure and sampling, and fast contour detection. In combination with an automated imaging system it is possible to measure thousands of cells in one day, with no user input. We validate this new pipeline by chemical modification of RBC mechanics, and by comparison with simulated fluctuations including each confounding effect. The methods and codes for this robust flickering analysis will allow consistent measurements across labs.