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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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