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Not sure this is the answer to your issue, but we had something a bit similar once with one of our users. In their case, they were using a mito-tracker dye to look at mitochondria. Signal kept going up the more we imaged. Don't remember if we were doing FRAP or just imaging. After much head scratching, I told them to do a dilution of their label. It turns out they were using about 1000x times more than what they needed and my explanation is that the dye was self-quenching; by "bleaching" it,  we were somehow reducing the  quenching and getting an increase in signal. When they used a 1000 fold dilution ( or at least a few hundred fold), the signal was still high or higher than when they were overloading, and no increase was seen during imaging. Doing a dilution series with GFP is not quite as trivial, but I wonder if they are seeing similar issues due to gross overexpression of their GFP?

Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109

http://www.fredhutch.org





On Sep 19, 2014, at 1:43 PM, Wendy Salmon wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
> 
> Hello all, 
> 
> I am hoping that the vast experience and expertise here might have an answer to a strange phenomenon one of my users is seeing with FRAP on my Zeiss 710. She is FRAP-ing ~2um diameter region in cells expressing GFP-fusion proteins with close to diffusion rates. 
> 
> After the regional bleach event and diffusion recovery, many of the cells exhibit a significant increase in total intensity over the starting intensity. Some of the time-intensity plots make it look like the recovery is 150% or more! Imaging cells without the bleach event never results in this signal increase. 
> 
> Has anyone seen this before and have suggestions on what might be going on? 
> 
> The FRAP settings are 40x/1.3NA oil objective with 100% power of the 488 Argon at scan speed of 2 for 1 iteration using the Zoom Bleach function over ~2um of the cell--this yields ~80% bleaching. For imaging, we are using very low laser excitation, finding cells with transmitted light and are not using the live image for image focus--typical live imaging stuff. For the cells that do not get extra bright after bleaching, the recovery rates are similar to published results. 
> 
> Ideas I have come up with are: 
> - Unquenching upon bleaching: If this were the case, I would expect that lowering the power of the FRAP settings would result in a brightening of the FRAP region (instead of bleaching all molecules in the volume, only some would bleach and create regional unquenching), not just reduction in photobleaching. 
> - Phototoxicity: Reducing the FRAP laser power or iterations or increasing scan speed only results in reduced bleaching, so I'm not sure how best to control for this. Perhaps adding Oxyrase or Trolox to give the cells some help against the ROS? 
> 
> Many thanks! 
> Wendy 
> 
> ~~~~~~~~~~~~~~~~~~~~~~~ 
> Wendy Salmon 
> Light Microscopy Specialist 
> Whitehead Institute for Biomedical Research 
> W.M. Keck Imaging Facility 
> 9 Cambridge Center, Rm 447 
> Cambridge, MA 02142 
> e: [log in to unmask] 
> w: http://staffa.wi.mit.edu/microscopy/