Re: Photobleach (FRAP) problem in Zeiss 510Meta

Posted by Claire Brown on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5043727.html

I would suggest you fix some cells and then test out your bleaching protocol. In my experience with live cells much of the recovery is faster than you would think. When I first started bleaching focal adhesions I didn't think I was bleaching but it was just recovering too fast.

I usually use a long box only 20 or so pixels high. As others have mentioned the confocal scans the entire line anyway and just turns on the observation or bleach intensity in the ROIs so you may as well make the region long in y and get lots of data. With the box small you should have a fast enough scan time to see the bleach.

Also take care with live cells not to bleach too much. When you use successive bleaches (like 50) you deplete a whole region of the cell and your kinetics may be dominated by diffusion back into the ROI rather than the dynamics in the ROI itself. This much laser light can also cause phototoxicity to the cells. I usually recommend one bleach scan if you can manage and never more than 10. Remember the shape of the recovery curve should be the same whether you bleach 20% or 100% of the fluorescence (assuming you are not depleting the entire region of fluorescence when you bleach more). So less is better.

Claire