Re: Photobleach (FRAP) problem in Zeiss 510Meta

Posted by G.Liu on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Photobleach-FRAP-problem-in-Zeiss-510Meta-tp5038048p5051254.html

Dear All,

Thanks you very much for all of your input (comments, suggestions, protocols etc ) in regard to the photobleaching in our Zeiss510. The good news is that we got some progress on this but bad news is that a new question needs to be addressed: the movement of the bleaching subject/organelle.

We are using transiently GFP-expressed tobacco leaves and want to bleach whole or partial nucleus. However the nucleus seems to move at lease about 1-2um in a 5min period. we tried to use larger pinhole and larger ROI but the plane of focus is still an issue (so only saw partial bleach). Zeiss specialist suggest to stick the leaves on the slide with krazy glue and surrounded by something to avoid the leave floating/vibrating (we are using inverted microscope). We tried but the movement was still noticeable. Have some of you meet a similar problem and how did it solved?

Meantime I am want to install a macro that could track the cell movement (developed by Ellenberg). Any experience on it?

Thank you in advance.