http://confocal-microscopy-list.275.s1.nabble.com/Bleaching-of-background-tp7578651p7578652.html
unfortunately I do not have an explanation for your background. Nevertheless I just wanted to make some remarks regarding your conclusions:
I personally would consider 750 ms-2.5 s as a long exposure times. Therefore I don't think that bleaching can be ruled out.
But you can easily test it experimentally. Just image with a higher temporal resolution (e.g. image every 5 minutes) and check whether the drop in intensity is faster or as fast as before. In the latter case you can be sure that bleaching is not an issue.
In the link below they describe that collagen itself is auto-fluorescent. So maybe the background is coming from the matrix. And as the matrix protein is probably higher concentrated (at least locally) as the GFP is could make an considerable contribution. This auto fluorescence can be increased due to an leakage of your filter-cube in the UV. It is known that a small amount of UV light is always present (therefore cells tend to be longer happy when the are illuminated with LED's).
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:
[hidden email]] On Behalf Of SANJUAN
> SAMARRA, XAVIER
> Sent: lundi 16 juillet 2012 12:26
> To:
[hidden email]
> Subject: Bleaching of background?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> 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