Structural analysis of red blood cell membrane with an atomic force microscope.

Abstract

To evaluate the usefulness of the atomic force microscope (AFM) for structural analysis of biomedical samples and to determine suitable sample preparation methods for AFM observation, the membrane of human erythrocytes prepared by various methods for electron microscopy was examined by the AFM. Strand-like elevations with 20-50 nm in width, 30-80 nm in length and 3-5 nm in height were observed, which formed networks composed of squares, pentagons and hexagons on the cytoplasmic or back surface of the erythrocyte membrane. Using colloidal gold labelled antibody, this network was found to contain spectrin molecules. Therefore it was very likely that the undercoat molecules of the plasma membrane were imaged by AFM. A large number of gentle elevations 300-400 nm in diameter and 2 nm in height were found to be distributed uniformly on the extracellular or true surface of intact erythrocyte, presumably reflecting the presence of undercoat membrane skeleton on the cytoplasmic surface. However, no structure that seemed to be derived from glycocalyces was discernible on the true surface. Structure corresponding to the unit membrane or lipid bilayer structure observable by electron microscopy was not demonstrated in the cross-section of the membrane. In freeze-fractured samples, a large number of small particles that corresponded to the intramembranous particle were also demonstrated on the membrane halves. Since AFM allows depiction of the fine structures of biological samples with very simple sample processing at a resolution comparable to or exceeding that of SEM, imaging technology using AFM can be applied to obtain biomedical information. However, several problems have to be solved in future development of the equipment.