Quantitative analysis of intramembrane particle (IMP) distribution on biomembranes after freeze-fracture preparation by a computer-based technique.

Abstract

An improved method for the quantitative analysis of intramembrane particles (IMPs) on freeze-fracture faces of biomembranes is described. The analysis provides, first, the number of IMPs per 1 micrometer2, second, the coefficient of dispersion (CD) of unweighted and weighted (i.e. the finite size is taken into consideration) IMP distribution, and, third, the diameters of IMPs. Additional data for further statistical evaluation are also derived. These data make it possible to numerically describe and characterize fracture faces of a membrane in a defined physiological state. They allow for comparison of normal membranes with experimentally induced alterations of the same specimen in other experiments, and for observation of differences between normal and malignant cells. Furthermore, such an analysis provides the basis for a dynamic interpretation of biomembrane components seen on the static freeze-fracture micrographs. With this numerical analysis, descriptive terms for IMP distribution such as 'random', 'aggregated' or 'clustered', which cannot be evaluated or verified, are avoided. The method, based on the Poisson statistic which provides a means for the description of different particle density distributions, is described. The validity of the method is demonstrated on artificially generated pictures and by an analysis of erythrocyte membranes before and after spectrin depletion.