We have deisgned and developed a new probe which links Sulfo-Cy5 to a membrane protein. This gives great confocal imaging however when we move to TIRF we see extensive bleaching of the fluorophore. The speed of the bleaching makes it difficult to obtain decent data for analysis. We have tried to offset this by reducing laser power and exposure time but to no avail. From a literature search I can’t find evidence of bleaching to this extent. Has anyone had any experience in this? Why does TIRF imaging bleach our probe so extensively?
We may try other fluorescent probes (obviously there is a time element here in development).
Thanks for any thoughts!