Straightforward (but expensive) way to do this would be to install the second CCD (or EMCCD) camera on your microscope and acquire images in two channels simultaneously. Olympus IX71 microscope can support two cameras and two optical paths with separate fluorescence cubes.

Another idea is very simple, but might work only if your fluorescent signals are very bright. Just select the barrier filter so you can see both fluorescent colors simultaneously and use regular color digital camera. This might also work with low intensity signals if they are spatially stable. Just increase the exposure time on you camera in this case.

Best,

Anatoly

Thanks Anatoly. Will look into that. 

Dear Anil,

I have found this article by Bolte et al. really helpful when I started with colocalization studys. http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2006.01706.x/pdf

Best Soenke

Thanks Sönke.

I have used a split image with one camera as well. Then I used the Cairn image tool plug-in for Image J to align the two images. There are also plug-ins to analyze colocalization.  Image J is free image analysis software that is available online. I agree with Anatole about needing bright signals and will add that it is much better if they have similar intensities.

 Thanks Peter and Teresa. Will look into these options. 

You're welcome. Best wishes. Teresa