Roughness of the plasma membrane as an independent morphological parameter to study RBCs: a quantitative atomic force microscopy investigation.

A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane-skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.