Correction of image instability in confocal microscopy using image realignment. Effects on the analysis of intracellular calcium

Using confocal microscopy, we have examined the increases in Ca(2+)](i) evoked by sodium channel toxins in cells labelled with the fluorescent dye INDO-1. We describe a new image analysis method that improves the detection of region-specific, toxin-induced patterns of change of intracellular calcium. This method is based on correction of global image motion followed by calculation of the strength of correlation between calcium changes in "seed" or reference pixels chosen to represent different regions of cells and those in other regions of the image. When the selected "seed" pixel was chosen to be in either varicosities or neurites, correlations were detected in the same regions of other cells as well as in the soma, indicating specific but spatially distinct patterns of behaviour. Control images (without changes in Ca(2+)](i)) did not reveal significant interpixel correlations. The ability to recognize correlated patterns of calcium change in different regions of cells was greatly improved by correction for global motion.