Deconvolution Combined with Digital Colocalization Analysis to Study the Spatial Distribution of Tight and Adherens Junction Proteins

Abstract

The relative spatial distribution of proteins was investigated with immunofluorescent methods by confocal laser scanning microscopy and digital image restoration. For confocal data sets recorded with a voxel dimension of 50 × 50 × 150 nm noise and blur can be decreased and the resolution in the z-axis increased by applying the maximum likelihood estimation algorithm of the Huygens software. This approach was successfully applied to the study of tight and adherens junctions in relation to the actin cytoskeleton in Madin Darby Canine Kidney cells. Colocalization analysis was done for pairs of probes using a histogram-based method. F-actin, occludin, zonula occludens 1, and E-cadherin were included in the study. Double-labeled preparations were used. The combination of deconvolution with the colocalization of confocal data sets offers a powerful tool to investigate the spatial arrangement of proteins.