Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Background-fluorescence-problem-tp7579031p7579038.html
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Hi Simon,
likely riboflavin and possibly other flavins. See
http://www.evrogen.com/products/medium_DMEM_gfp/medium_DMEM_gfp.shtml
and the Bogdanov et al paper referenced at the bottom of the page;
* Bogdanov AM, Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov
S, Lukyanov KA. Cell culture medium affects GFP photostability: a
solution. Nat Methods. 2009; 6 (12):859-60. / pmid: 19935837
<
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=19935837&dopt=Abstract>
Their solution: incubate cells in miedia without (or with low, if
needed) riboflavin for a day.
As a bonus, riboflavin quenches (FRET?) and/or transiently photoconverts
GFP to red fluorescence (might be mostly dark states):
Condensed mitotic chromosome structure at nanometer resolution using
PALM and EGFP- histones. </pubmed/20856676>* Matsuda* A, Shao L,
Boulanger J, Kervrann C, Carlton PM, Kner P, Agard D, *Sedat* JW. PLoS
One. 2010 Sep 15;5(9):e12768. PMID: 20856676
If you contact Essen Biosciences, they will (hopefully) give you a copy
of their application note on the concentrations of riboflavin in many
culture media and correlation with fluorescence of those media. Speaking
of Essen - they finally introduced a dual green+red fluorescence Incucyte.
Enjoy,
George
On 9/13/2012 11:04 AM, Simon Walker wrote:
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>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Dear List,
> We are imaging very weakly fluorescent live cells (expressing GFP) on a wide-
> field system and having issues with a source of background fluorescence.
> When we look at our cells under epi-illumination we see a rapid drop in a weak
> background signal (not where the cells are) that fully recovers over a ~10 s
> period after the illumination light is switched off. Our experiments require the
> use of DMEM as the imaging medium and this is the likely cause of problem. It
> appears that something in the medium is sticking to the coverglass. It's not
> phenol red as the effect is seen with both phenol red-containing and phenol-
> red-free DMEM. Does anyone know what else it could be? Has anyone else
> seen anything similar? We're wondering if it could be riboflavin which is in the
> DMEM we're using. Would this stick to glass?
>
> I've seen that Life Technologies now market a substance that allegedly
> surpresses background fluorescence in DMEM:
>
http://products.invitrogen.com/ivgn/product/R37603> Has anyone tried this? Does anyone know how it works?
>
> Thanks,
> Simon
>
>