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Hallo,
a recent thread "Increase in cell brightness after FRAP" reminded me of a
strange recovery behavior I have seen in my datasets.
I would appreciate opinions/explanations regarding this mystery.
A cytoskeletal protein localized in plant chloroplasts is tagged either with
GFP or mYFP.
The recovery is slow, half times are in hundreds of seconds.
Unexpectedly, in many datasets the fluorescence in the bleach ROI remains
flat or even continues to decline for many tens of seconds before the
recovery can be observed.
This is after background subtraction, correction for photobleaching by
imaging, and normalization.
I am imaging with a 60/1.2 water immersion objective. Pixel size 0.1 um.
Bleach ROI is a 2 x 2 micron square
excitation and bleaching with 488nm laser
I do not think it is a photoswitching phenomenon, since that would happen
on a much shorter time scale. Furthermore, the same fluorescent tags
used with a closely related cytoskeletal protein in the same chloroplast
compartment showed normal recovery curves.
I thought perhaps there could be transient binding (the protein is known
to interact with a membrane protein in the chloroplast envelope).
or the photobleached fluorescent protein is more sensitive to the post-
bleach imaging laser, thus my correction for fluorescence decay is nor right.
This strange behavior happens predominantly at higher expression levels.
Thanks in advance!
Stan Vitha
Microscopy and Imaging Center
Texas A&M University
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