Posted by
xavier Sanjuan on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Bleaching-of-background-tp7578651.html
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Dear listers,
One of our users is making experiments to measure GFP signal in a cell
culture. Background fluorescence is quite strong and GFP signal is just
slightly a bit stronger than that. The thing we do not have an explanation
for is we are detecting an exponential decrease in background signal
intensity. Briefly the details of the experiment are:
Cells are cultured in Lab-Tek chambers in a collagen matrix (prepared with
phenol red from MEM 10x medium) and (in their initial experiments) kept in
medium with FBS + phenol red. Plates were imaged in a Zeiss Cell Observer
using a 20x dry objective and, as GFP signal is really low, an EMCCD
camera. Images were obtained every 30 min during 24h and exposure time was
between 750 ms and 2.5 s depending on the experiment.
As mentioned, under these conditions background signal is quite high but
over time we detect an exponential decrease in its intensity. As phenol red
is a known source of autofluorescence, we advised him to use medium without
it, but in these new experiments we observed the same phenomena. Wells with
only collagen + medium also showed that issue. He will try to prepare the
collagen also without phenol red just to totally discard this component as
a background source, but we still have the issue of "bleaching" of the
background. I don't expect bleaching to be an issue with that modest amount
of exposure, at least for GFP; and we all know autofluorescent components
tend to be more resistant to photobleaching than the labels we introduce in
our samples...
Anyone has an explanation for this exponential decrease in background
intensity then?
Thank you very much for your help,
Xavi.
___________________________________
*Xavier Sanjuan**
*Advanced Light Microscopy Unit
Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88
08003 Barcelona - Spain
Tel: + 34 93 316 0206 (ext 1206 dins el PRBB)
Fax: + 34 93 316 09 01
E-mail:
[hidden email]
Web:
http://pasteur.crg.es/portal/page/portal/Internet/03_CORES/Core_Facilities/Advanced%20Light%20Microscopy%20Unit