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Colocalization
Left: The image shows an Alzheimer's disease plaque (red) surrounded by active microglial cells (green). Right: Volocity was used to generate a positive (yellow) and a negative (purple) PDM channel. This gives a clear visual display of the areas and the degree of positive and negative correlation. Image courtesy of Professor Elizabeth Head, Sanders-Brown Center on Aging, University of Kentucky.
Microscopists often try to demonstrate relationships or associations between two molecules of interest. This work involves determining whether two fluorescent labels occur in the same spatial location, i.e. are colocalized. Overlaying or merging fluorescent images gives you an indication of potential colocalization but it is not conclusive and can sometimes be misleading. The latest advances in imaging software can now provide more accurate analysis in this essential imaging application.
Traditionally, software is used to create a merged view of the two channels as a means of demonstrating such a relationship or association. Whilst this can give a quick indication of where colocalization occurs, it is not a quantitative method. The technique does not address whether two proteins vary in synchrony, which is needed to give confidence that colocalization is truly occurring. To do this accurately the intensities of the original images must be broadly similar. All too often scientists rely on their observations and visual interpretations which can be very poor at detecting pixel intensity variations in two overlay channels. PerkinElmer’s solutions overcome these problems and can provide accurate quantitative measures of colocalization.