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Dear George,

I heartily applaud your concerted efforts and philosophy here!!!

Keep up the good work!

Dan

Daniel J. White Ph.D.
Max Planck Institute - CBG
Light Microscopy Facility
and Image Processing Facility.
Dresden, Germany.

Fiji is just ImageJ (batteries included)
http://pacific.mpi-cbg.de

BioImageXD
http://www.bioimagexd.net

http://www.chalkie.org.uk

Mobile +49 15114966933
Office +49 351 210 2627

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On 03.10.2010, at 07:01, CONFOCALMICROSCOPY automatic digest system <[log in to unmask] 
 > wrote:

> There is 1 message totalling 152 lines in this issue.
>
> Topics of the day:
>
>  1. Reduced CFP and YFP fluorescence
>
> ----------------------------------------------------------------------
>
> Date:    Sat, 2 Oct 2010 09:15:51 -0400
> From:    George McNamara <[log in to unmask]>
> Subject: Re: Reduced CFP and YFP fluorescence
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Hi Pablo,
>
> Could be poor folding. That is, at the single molecule level, a
> correctly folded FP may produce the same number of counts per second
> (photons) for a given excitation level as a free FP. Typically tested
> by increasing linker length on both ends of the FP. Using a
> Superfolder FP (they are available in several colors), plus longer
> linkers, may be useful. Another tool is to add a another FP
> (different color, matures well), at a free end - preferably on the
> extracellular side (in your case) to avoid FRET.
>
> At a trivial level, since you already have the constructs, growing
> your cells at lower temperature for a day or more, for example, 30 C,
> may enable better maturation/folding.
>
> Plenty of recent reviews on FP's - see especially:
>
>
> <http://www.ncbi.nlm.nih.gov/sites//pubmed/20664080>Fluorescent
> proteins and their applications in imaging living cells and tissues.
>
> Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA.
>
> Physiol Rev. 2010 Jul;90(3):1103-63. Review.PMID: 20664080
>
> Carl and I also have one:
>
> <http://www.formatex.org/microscopy3/papers.htm>Modern Research and
> Educational Topics on Microscopy - Content
> G. McNamara and C.A. Boswell.
> A Thousand Proteins of Light: 15 Years of Advances in Fluorescent  
> Proteins
> www.formatex.org/microscopy3/papers.htm
> (article and Excel file is about 1/4 way down ... search the web page
> for Boswell)
>
> spectra for most (older) FPs are in the xlsx file inside the ZIP  
> file at
> http://sylvester.org/research/shared-resources/laboratory-resources/analytical-imaging-core-facility/analytical-imaging-core-facility-links-forms/pubspectra-data
>
> Speaking of PubSpectra ... Everyone reading this - researchers or
> vendors - who have spectra you want to add to PubSpectra, please
> contact me directly.
>
> I am especially interested in objective lens transmission curves
> (vendors), LED and other novel light sources, filters, filter sets,
> internal filters (ex. the filters inside the LSM710/LSM780, SP5,
> SP5-II), multiphoton excitation (I recently organized the Xu/Webb
> Cornell data but not yet posted online), new FPs, new organic dyes,
> new QDots, new other Dots, photonconvertible/switchable/photo-etc
> before and after, SHG spectra (see recent PNAS paper from Scott
> Fraser's lab), STED depletion spectra. However, I want data, ideally
> in 1 nm intervals in Excel (and best of all organized as in
> PubSpectra), since un-scanning graphs has gotten old.
>
> I am happy to add other spectra to Pubspectra, and can always put
> other Excel files in the ZIP file. One could be Raman spectra. Leica
> CW-STED uses a 592 nm laser. The anti-Raman Stokes shifts for H2O
> Raman lines are 508 nm for 3600 cm-1 and 502 nm for 3300 cm-1 (I've
> never been sure which or both of these to use). I am also curious
> what Raman shifts would be useful to have for other mounting media,
> such as glycerol, thiodiethanol, Mowiol/polyvinyl alcohol, and
> additives such as the STED ROXS reagents (Kasper et al 2010 Small,
> PubMed 20521266).
>
> PubSpectra is data - data are facts, facts are not copyrightable (at
> least in the USA, which has a much more enlightened laws than the
> E.U.). PubSpectra is and always will be free to use. Zeiss already
> uses it for "Smart Setup" inside ZEN. I encourage all other vendors
> to incorporate PubSpectra into their software to make the spectra
> easy to find and graph.
>
> Enjoy,
>
> George
>
>
>
>
> At 10:44 PM 9/26/2010, you wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear List Members,
>>
>> I am working with 7 trans-membrane domain receptors. These are not
>> GPCRs. I have made chimeras by inserting CFP and YFP molecules at the
>> N- and C-terminus and in the intra-cellular loops similarly to what
>> other people have done previously (Villardaga, 2003) to be able to
>> detect conformational changes with FRET.
>>
>> I have noticed that when I insert CFP or YFP in the intra-cellular
>> loops the fluorescence intensity of both is reduced when compared to
>> N-terminus or C-terminus. I have shown by Western blot that the
>> expression levels is similar, therefore the fluorescent proteins have
>> lower fluorescent intensity.
>>
>> Does anyone have an idea why this could be happening? Could the
>> structure of the fluorescent proteins be affected by being held  
>> within
>> two trans-membrane domains? Or is it because the molecule is not free
>> to rotate?
>>
>> Any help would be appreciated.
>>
>> Kind regards,
>> Pablo
>>
>> --
>> Pablo German
>> PhD Candidate
>>
>> Plant and Food Research
>> Private Bag 92169
>> Auckland Mail Centre
>> Auckland 1142
>> New Zealand
>> DDI: (09) 925-7107
>> Mobile: 0210459406
>
>
>
>
>
>
>
> George McNamara, Ph.D.
> Image Core Manager
> Analytical Imaging Core Facility
> University of Miami, Miller School of Medicine
> Miami, FL 33136
> [log in to unmask]
> [log in to unmask]
> 305-243-8436 office
> http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
> http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+
> spectra .xlsx file is in the zip file)
> http://home.earthlink.net/~geomcnamara
>
> ------------------------------
>
> End of CONFOCALMICROSCOPY Digest - 1 Oct 2010 to 2 Oct 2010 (#2010-32)
> **********************************************************************