Did you try more than one widefield microskope? If not, think about chromatic shift. You should chrck this by using two structures where you know they colocalize as a control.

Best,

GAD

I agree with Guido. Check if the objective you use in the widefield microscope is corrected for the chromatic aberration.

Alternatively if you can exchange the objectives between microscopes try this one on the confocal.

Good luck,

Manel.

If you see coloc under CLSM and not under widefield, it smells like bleedthrough. This is very common when two colors are acquired simultaneously. Try sequential acquisition