Posted by
Mike Ignatius-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Background-fluorescence-problem-tp7579031p7579043.html
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This issue has been discussed several times on the listserve - including
this commentary back in 2006 from Gert van Cappellen, which parallels what
George and others are saying and publishing:
"Regarding the autofluorescence of the medium . To our experience riboflavin
free medium helps more than phenol-red free medium. I've started out using
completely phenol red-free DMEM (you can get that from Gibco) to reduce
background fluorescence, but it didn't help at all. After that, I've spent
some time on a fluorometer to check out all the components of our culture
media and in the case of DMEM, riboflavin caused more than 95% of the
fluorescence! Phenol-red itself is NOT significantly fluorescent in the
wavelength range relevant to GFP."
In fact there is good evidence that Phenol Red is a good quencher, including
the flavins and whatever green signal one is trying to detect!
Lelong and Rebel raise the additional concern that bicarbonate based buffers
can reach pH 8.5 in an hour if not maintained in CO2.
Lelong IH, Rebel G. (1998) "pH drift of "physiological buffers" and culture
media used for cell incubation during in vitro studies." J Pharmacol Toxicol
Methods 39(4):203-10
For applications where complete media is not essential (sorry Dr. Walker),
Marker Gene Technologies now offers a HEPES based buffer for imaging that:
I) provides minimal essential nutrients to keep cells metabolically
active with no autofluorescence.
II) contains reagents (and shipped in a brown bottle) to avoid the
toxicity associated with HEPES exposure to light
III) has a mild anti-oxidant for signal preservation.
IV) maintains proper pH and osmolarity for hours without CO2.
Our PIS describes the product it in more detail @
http://www.markergene.com/ProductDetails.php/M1898Mike
Mike Ignatius, Ph.D.
[hidden email]
Marker Gene Technologies Inc.
1850 Millrace Drive
Eugene, Oregon, USA 97403
541 342 3760
markergene.com
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On
Behalf Of George McNamara
Sent: Thursday, September 13, 2012 5:57 PM
To:
[hidden email]
Subject: Re: Background fluorescence problem
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Hi Simon,
likely riboflavin and possibly other flavins. See
http://www.evrogen.com/products/medium_DMEM_gfp/medium_DMEM_gfp.shtmland 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:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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
>
>