High Resolution Cryo-Electron Microscopy Sheds A New Light Upon Biomembrane Architecture

Abstract

Cryo-electron microscopy, together with the rapid cryo-fixation technique, has made it possible to observe chemically unfixed unstained specimens under a transmission electron microscope. Aoki et al. constructed a new cryo-stage in order to reduce irradiation damage upon biological specimens further by cooling them with liquid helium. It was also designed to eliminate shaking of specimens as much as possible by the sophisticated mounting and the refined cooling mechanism of the stage. The stage has proved to achieve a higher contrast and higher resolution of images than a commonly-used cryo-stage cooled solely by liquid nitrogen does. This high resolution cryo-electron microscope (HiRCEM) was originally developed aiming at the improvement of resolution in structural analysis of proteins or nucleic acids, but it has turned out to be quite useful for detecting the architecture of biomembranes as well. This is due to the existence of phosphorus atoms of phospholipids which are heavier than C, H, O, N atoms abundant within biomembranes, and also due to the great optical depth of the membrane where it is aligned parallel to the electron beams.We have examined several kinds of biomembranes as well as the synthesized liposomes under HiRCEM. Among them were the plasma membrane of human erythrocytes (ghosts), the nuclear membrane of chicken erythrocytes, the endoplasmic reticular membrane of rat nerve cells, the chromatophore membrane of a photosynthetic bacterium Chromatium vinosum , the thylakoid membrane of spinach chloroplasts and the envelope of influenza type A virus (PR8 & X31). Liposomes we employed were synthesized from the 2:1 mixture of phosphatidylcholine and cholesterol.