Cell Topography Correction in Colocalization Analysis

Abstract

Colocalization analysis is the study of the distributions of species with fluorescence microscopy. By estimating the spatial distribution of the fluoresence emission from two types of molecules, colocalization analysis is used as a method to help in the understanding of processes and interactions between molecules both within cells and with their environment. One of the basic assumptions of colocalization analysis today is that the species of interest are spatially distributed on a uniform surface, their only interaction being with each other. The assumption of a uniform surface however, does not always hold. The plasma membrane for example, is not flat but can be convoluted, folded and deformed in a large number of ways, causing the assumption of a flat surface to not be applicable when studying molecules present in the membrane of a cell. With aim to improve the result of colocalization analysis, we present a method of intensity normalization by weighting the observed intensities of the species of interest by the membrane distribution. To study the effect of background normalization on colocalization analysis, simulations were conducted on non-uniform surfaces where the Pearson correlation coefficient and Spearman rank correlation coefficient were used to estimate the amount of colocalization before and after background normalization. Based on the simulations, together with results from analysis of experimental images using confocal laser scanning microscopy, we present a proof of concept for the effect of background normalization on colocalization analysis.