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September 2014

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From:
Stanislav Vitha <[log in to unmask]>
Reply To:
Confocal Microscopy List <[log in to unmask]>
Date:
Mon, 22 Sep 2014 15:28:38 -0500
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perhaps the culture medium plays a role? 
see for instance Bogdanov, A. M., Kudryavtseva, E. I., and Lukyanov, K. A. 
(2012) Anti-fading media for live cell GFP imaging, PLoS One 7, e53004.

 
I had a similar experience when doing FRAP of GFP or YFP tagged 
cytoskeletal proteins in plant chloroplasts in intact leaf tissue.  Bleaching 
with a 405 nm laser bleached the chlorophyll but caused a bright spot in 
the green channel, or a donut-shaped spot (dark spot surrounded by 
brighter area.
I attributed that to photoswitching of the fluorescent protein from the dark 
state to the fluorescent state.  the darker center of the donut was where 
the actual photobleaching occurred.

It seems many fluorescent proteins can be photoswitched, at least few 
times.  Here is an example of mOrange2 photoswitching - cytoskeletal 
protein expressed in yeast, alternating 543nm excitation (5 frames) and 
405 nm (5 frames). 
http://i1219.photobucket.com/albums/dd436/bohemyan/MIC/Pom_2013-
03-mOrange2-photoswitch.png

Stan Vitha
Microscopy and Imaging Center
Texas A&M Univeristy

  

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