http://confocal-microscopy-list.275.s1.nabble.com/Background-fluorescence-problem-tp7579031p7579045.html
processes in living cells. The standard genetically encoded FRET pair
photoconversion. We engineered two fluorescent proteins, Clover and
yet described. Replacement of CFP and YFP with these two proteins in
emission ratio changes. These improvements enhance detection of
> *****
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>
> What is Clover GFP? I can't find much information about it on Google.
>
> Thanks,
> Kurt
>
> On 9/14/2012 3:49 AM, George McNamara wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
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>>
>> Hi Simon,
>>
>> Your cells might not need the 100x excess of riboflavin present in
>> "standard" DMEM, your background could be reduced. The Essen tech
>> note I mentioned lists:
>> DMEM 0.4 mg/L riboflavin ... 43.6 units fluorescence
>> Eagles MEM 0.1 mg/mL ... 12.9
>> F12K 0.04 ... 5.4
>> EBM 0.004 ... 3.7
>> Riboflavin (alone) 0.4 ... 58.7 (perhaps suggesting that
>> culture media quenches riboflavin or it gets converted in part to
>> something less fluorescent?)
>> Contact Essen if you want the entire tech note.
>>
>>
>> If you absolutely require a green fluorescent protein, spend the time
>> to switch to the new Clover or "V6" from Steven Vogel (available from
>> addgene.org as VVVVVV).
>> If you do not need green, switch to tdTomato or the new mRuby2.
>>
>>
>>
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>> Nat Methods. <#> 2012 Sep 9. doi: 10.1038/nmeth.2171. [Epub ahead of
>> print]
>>
>>
>> Improving FRET dynamic range with bright green and red fluorescent
>> proteins.
>>
>> Lam AJ
>> </pubmed?term=Lam%20AJ%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, St-Pierre
>> F
>> </pubmed?term=St-Pierre%20F%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>,
>> Gong Y
>> </pubmed?term=Gong%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, Marshall
>> JD
>> </pubmed?term=Marshall%20JD%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>,
>> Cranfill PJ
>> </pubmed?term=Cranfill%20PJ%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>,
>> Baird MA
>> </pubmed?term=Baird%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>,
>> McKeown MR
>> </pubmed?term=McKeown%20MR%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>,
>> Wiedenmann J
>> </pubmed?term=Wiedenmann%20J%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>,
>> Davidson MW
>> </pubmed?term=Davidson%20MW%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>,
>> Schnitzer MJ
>> </pubmed?term=Schnitzer%20MJ%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>,
>> Tsien RY
>> </pubmed?term=Tsien%20RY%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>,
>> Lin MZ
>> </pubmed?term=Lin%20MZ%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>.
>>
>>
>> Source
>>
>> 1] Department of Bioengineering, Stanford University, Stanford,
>> California, USA. [2] Department of Pediatrics, Stanford University,
>> Stanford, California, USA.
>>
>>
>> Abstract
>>
>> A variety of genetically encoded reporters use changes in
>> fluorescence resonance energy transfer (FRET) to report on
>> biochemical processes in living cells. The standard genetically
>> encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP
>> reporters suffer from low FRET dynamic range, phototoxicity from the
>> CFP excitation light and complex photokinetic events such as
>> reversible photobleaching and photoconversion. We engineered two
>> fluorescent proteins,* Clover and mRuby2*, which are the brightest
>> green and red fluorescent proteins to date and have the highest
>> Förster radius of any ratiometric FRET pair yet described.
>> Replacement of CFP and YFP with these two proteins in reporters of
>> kinase activity, small GTPase activity and transmembrane voltage
>> significantly improves photostability, FRET dynamic range and
>> emission ratio changes. These improvements enhance detection of
>> transient biochemical events such as neuronal action-potential firing
>> and RhoA activation in growth cones.
>>
>> PMID:
>> 22961245
>>
>>
>> PLoS One. <#> 2012;7(5):e38209. Epub 2012 May 30.
>>
>>
>> Fluorescence polarization and fluctuation analysis monitors subunit
>> proximity, stoichiometry, and protein complex hydrodynamics.
>>
>> Nguyen TA
>> </pubmed?term=Nguyen%20TA%5BAuthor%5D&cauthor=true&cauthor_uid=22666486>,
>> Sarkar P
>> </pubmed?term=Sarkar%20P%5BAuthor%5D&cauthor=true&cauthor_uid=22666486>,
>> Veetil JV
>> </pubmed?term=Veetil%20JV%5BAuthor%5D&cauthor=true&cauthor_uid=22666486>,
>> Koushik SV
>> </pubmed?term=Koushik%20SV%5BAuthor%5D&cauthor=true&cauthor_uid=22666486>,
>> Vogel SS
>> </pubmed?term=Vogel%20SS%5BAuthor%5D&cauthor=true&cauthor_uid=22666486>.
>>
>>
>> Source
>>
>> Section on Cellular Biophotonics, Laboratory of Molecular Physiology,
>> National Institute on Alcohol Abuse and Alcoholism, National
>> Institutes of Health, Rockville, Maryland, United States of America.
>>
>>
>> Abstract
>>
>> Förster resonance energy transfer (FRET) microscopy is frequently
>> used to study protein interactions and conformational changes in
>> living cells. The utility of FRET is limited by false positive and
>> negative signals. To overcome these limitations we have developed
>> Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid
>> single-molecule based method combining time-resolved fluorescence
>> anisotropy (homo-FRET) and fluorescence correlation spectroscopy.
>> Using FPFA, homo-FRET (a 1-10 nm proximity gauge), brightness (a
>> measure of the number of fluorescent subunits in a complex), and
>> correlation time (an attribute sensitive to the mass and shape of a
>> protein complex) can be simultaneously measured. These measurements
>> together rigorously constrain the interpretation of FRET signals.
>> Venus based control-constructs were used to validate FPFA. The
>> utility of FPFA was demonstrated by measuring in living cells the
>> number of subunits in the ?-isoform of Venus-tagged
>> calcium-calmodulin dependent protein kinase-II (CaMKII?) holoenzyme.
>> Brightness analysis revealed that the holoenzyme has, on average,
>> 11.9 ± 1.2 subunit, but values ranged from 10-14 in individual cells.
>> Homo-FRET analysis simultaneously detected that catalytic domains
>> were arranged as dimers in the dodecameric holoenzyme, and this
>> paired organization was confirmed by quantitative hetero-FRET
>> analysis. In freshly prepared cell homogenates FPFA detected only
>> 10.2 ± 1.3 subunits in the holoenzyme with values ranging from 9-12.
>> Despite the reduction in subunit number, catalytic domains were still
>> arranged as pairs in homogenates. Thus, FPFA suggests that while the
>> absolute number of subunits in an auto-inhibited holoenzyme might
>> vary from cell to cell, the organization of catalytic domains into
>> pairs is preserved.
>>
>> PMID:
>> 22666486
>>
>>
>> I am a bit disappointed Vogel's group did not go for V8 (a well known
>> drink) or V12 - the latter either as a polypeptide or with inducible
>> dimerization domain. V12 since the goal of this paper is to quantify
>> the number of subunits in CaMKIIalpha, which turns out to be 12 (+/-
>> a few) as described in
>>
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364239/figure/pone-0038209-g004/
>>
>>
>> On 9/14/2012 4:32 AM, simon walker wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>>> *****
>>>
>>> Thanks for the various responses. Yes, I'd seen the Bogdanov paper
>>> and the Evrogen medium and thought that might be worth a try. The
>>> problem we have is that for our assay the culture medium is
>>> absolutely critical (it's not just a case of keeping cells alive),
>>> so we can't use a minimal HEPES-based buffer. I am interested to
>>> know what is in the 'BackDrop' solution. We can't use it unless
>>> we're fairly confident it's not going to affect our assay.
>>> Simon
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List
>>> [mailto:
[hidden email]] On Behalf Of George McNamara
>>> Sent: 14 September 2012 01:57
>>> To:
[hidden email]
>>> Subject: Re: Background fluorescence problem
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>>> *****
>>>
>>> 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:
>>>> *****
>>>> 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
>>>>
>>>>
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>>
>>
>