Atomic Force Microscopy of Erythrocyte Cytoskeleton

Atomic force microscopy (AFM) has been used for investigate the erythrocyte cytoskeleton structure and its stability without removing the plasma membrane by detergent treatment and without extending or staining the cytoskeleton. A novel lectin attachment method was developed to immobilize cell membranes on glass surfaces for AFM imaging and a morphological image analysis protocol was built to quantify cytoskeleton structural change under different sample preparation conditions. Human healthy and pathological erythrocytes as well as murine mutant erythrocytes were examined with the developed method. An aggregation model was proposed to explain and quantify the observed morphological changes of the cytoskeleton. Also studied was the effect of free Ca2+ on the cytoskeleton structure and properties of healthy young erythrocytes. Finally, tapping-mode AFM was used to investigate cytoskeleton elastic properties. This thesis provides evidence to demonstrate that AFM combined with quantitative image analysis is a promising approach to connect biochemical and structural data for the study of cytoskeleton physical properties.