Quantitative picture analysis of freeze-fracture electron-micrographs.

A method of three-dimensional reconstruction of the surface profile of artificial and natural membranes from freeze quenched electron micrographs is presented. The direct relation between the Pt-layer thickness and the local orientation of the membrane allows a reconstruction of the surface. The efficiency of this method is demonstrated on the quantitative analysis of some fine structures. These essential results are: 1. In the low resolution observation structural elements of a yeast cell were quantitatively described, (i) The diameter of a yeast cell is determined (4.2 microgram). (ii) The cell wall thickness is measured (150 nm). (iii) The dimension of cell wall incapsulated vesicles is determined (60-80 nm). (iv) The damlike protrusion in the plasma membrane has a triangular cross section. The height is 23 nm and the half width 50 nm. The particle assembly in the damlike protrusion is in a crystalline state. The change in surface curvature is probable due to a phase separation of a biaxial cluster in an uniaxial membrane. (v) Membrane bound particles can be distinguished by their surface profiles. 2. The resolution of surface profiles is limited by the size of the platinum grain. An average procedure can lead to a resolution of 0.2 nm. This increase is resolution can be understood with the uncertainty relation: The uncertainty of the profile in one dimension times the uncertainty in the other dimension (averaging length) is the area of the platinum grain. The monolayer thickness of dipalmitoyl phosphatidyl choline and dimyristoyl choline are distinguishable 2.6 +/- 0.2 nm and 2.4 +/- 0.2 nm respectively. The surface profile of a two-dimensional crystal in the membrane of a yeast cell can be determined with high accuracy. The two profiles of the inner and outer monolayer do not fit exactly together. A part of the membrane bound particle is pulled out of the monolayer during the fracturing procedure. 3. The third part investigates special fluctuation of the surface. (i) The mixture of dipalmitoyl phosphatidyl choline and dioleyl phosphatidyl choline shows a periodic structure. The fluctuation besides this periodicity can be explained by a spinodal decomposition during cryofixation. (ii) The fluctuation of a periodic structure can also be induced by thermal motion. The fluctuation of dimyristol phosphatidyl choline quenched from a temperature between the pre- and maintransition determines only one kind of elastic constant. This curvature elastic constant is in the order of 10(-20) Joule. (iii) The fluctuation of the particle density can be related with the particle-particle compressibility. We choose the clusters induced by polylysine in a membrane with charged and uncharged lipids as particles. The compressibility is in the order of 10(-6) Newton/m which is comparable to those of a monolayer in a gaseous state.