Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi everyone!

i'm doing FRAP experiments on a Zeiss LSM510 meta and i wonder why the
following happens:
in the scan that immediately follows the bleach event i get an overall
drop in gfp-fluorescence within the entire field of view. there seems
to be no correlation with the distance from the bleach area. the very
surprising thing is now that the scan thereafter shows again recovery
of fluorescence.
anybody knows what is causing this? is the postbleach scan somehow
affected by the previous bleach?
note: this also happens on fixed cell preparations not only in living
samples.

question number 2:
what is the best way to correct for overall bleach-by-scans effects? to
me it appears as if regions with high initial fluorescence are much
more bleached by subsequent scans than ones with low fluorescence.
so how to respect this in the recovery kinetics of FRAP?
so far i just took the average loss-in-fluorescence of a neighbouring
region for correction.
should one determine this bleach function with respect to initial
fluorescence intensity and correct the recovery measurements with this
function?
is there a literature reference that explains how to do?

thanks a lot in advance
Tobias