Cytometry Lab

Colocalisation in Confocal Microscopy

A property determinable using confocal microscopy is the colocalisation of sub-cellular constituents and that can be done by attaching a differently coloured fluorescent tag to each. The colocalisation can be seen in a composite image; for example, where two constituents are separately labeled in fluorochromes depicted as red and green, areas of colocalisation will appear as yellow.

To extract a numeric measure, the first step may be to count the image pixels into a 2-dimensional histogram according to their red and green intensities. This count may be restricted to a selected region of interest (ROI) within the image.

High colocalisation would then be reflected by a large proportion of pixels plotting along the histogram diagonal. A rating of the colocalisation shown in the original image can then be obtained by setting a region of the histogram within which pixels are deemed to represent colocalised fluorochromes and counting the proportion of pixels therein. Areas of the original image in which these pixels appear can also be marked.

This process has been implemented as a simple plugin addition to the ImageJ image processing package. The results shown above have been obtained from this system. In keeping with the open source philosophy of ImageJ, the source code for the plugin is available HERE.