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... maybe one way to test that is to run a time series with higher than normal laser power (and accordingly lower gain) and see if intensity increases over time...


Julio 

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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/