A new method to measure bilayer thickness: cryo-electron microscopy of frozen hydrated liposomes and image simulation

A new method to measure the bilayer thickness of liposomes is described. Frozen hydrated liposomes composed of either phosphatidylcholine (PC) or PC containing 30 mol% cholesterol were observed in vitreous ice with a cryo-electron microscope, which enables high-resolution observation of frozen-hydrated unstained biological specimens. The examined PCs were di-(saturated acyl) PCs (di-C12:0 (DL), diC14:0 (DM), di-C16:0 (DP)), unsaturated acyl PCs (diC18:1 (DO), C16:0-C18:1 (PO)) and PC from a natural source (egg-yolk (EY) PC). Every liposome image displayed a pair of concentric circles indicating a different bilayer structure due to the acyl-chain species. The observation of the liposomes showed the following results; (1) liposomes including unsaturated acyl PC (DOPC, POPC and EYPC) displayed a smooth and regular image of the bilayer and sharp distributions of the bilayer thickness, while the images of those composed of di-(saturated acyl) PC (DLPC, DMPC and DPPC) showed irregular shapes and wide distributions of the bilayer thickness, (2) addition of 30 mol% cholesterol to the di-(saturated acyl) PCs dispelled the irregularity in the shape of the membrane and the distribution of the bilayer thickness, and (3) the mean bilayer thickness of the liposome composed of di-(saturated acyl) PCs increased corresponding to the carbon numbers of the acyl chains. The reliability of the observations and measurements was confirmed by coupling of the cryo-electron microscopy with image simulation using the MULTI SLICE computer program and the error estimation of +/- 0.63 A using images of crystalline chlorinated copper phthalocyanine.