Coherent 622 argon ion laser
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Glen MacDonald-2 |
Coherent 622 argon ion laser
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Monkey see, monkey do. Allen's offer has inspired me to do some surplus action. We've got a Coherent Enterprise 622 argon ion laser, 250 Watt, 351, 357, 363, 488, 514 lines, with recirculating water cooler and manuals. It came off of a Bio-Rad MRC-1024 that has been refitted for fiber coupled solid state lasers. It's been sitting in its shipping crate for 2 years. Anybody want it for the cost of shipping? Please contact me directly. Regards, Glen Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] ************************************************************************ ****** The box said "Requires WindowsXP or better", so I bought a Macintosh. ************************************************************************ ****** |
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John Oreopoulos |
FRET microscopy tips and references?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hello again Confocal listserver,
I've recently been assigned a to a collaborative project that involves trying to infer membrane protein dimerization by FRET microscopy. The proteins being imaged have been labeled with CFP and YFP. While I understand the basics of FRET - that when the two fluorochrome dipoles come close together the FRET efficiency, E, goes up and this can be monitored by various ratiometric imaging strategies - it's not clear to me how the E value can be used to quantitatively determine dimerization, however. I'm new to this type of imaging and was hoping someone out there could point me to a few references that explain how FRET imaging can be used to quantitatively determine oligimerization of proteins in general. Who was the first to do this experimentally? Is there a way to distinguish stoichiometry (dimers vs. trimers, etc)? I will be performing my experiments on a multi-laser line TIRF microscope fitted with an EMCCD camera and appropriate filter cubes. Of the FRET imaging strategies available, which one is the best? I've read a little bit about acceptor and donor photobleaching techniques, and these seem like the simplest ways to determine FRET, but how accurate are these methods compared to others? As usual, thanks to anyone who can enlighten me (no pun intended). John Oreopoulos, BSc, PhD Candidate University of Toronto Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging Tel: W:416-946-5022 |
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Rietdorf, Jens |
Re: FRET microscopy tips and references?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hello John,
a nice listing and comparision of 'quantification methods'
is in:
Zal
T, Gascoigne NR. Photobleaching-corrected FRET efficiency
imaging of live cells.
Biophys
J. 2004 Jun;86(6):3923-39. Erratum in: Biophys J.
2004 Oct;87(4):2915. nice
strategies for the quantification of c-yfp constructs is in
Cheers, jens
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos Sent: Mittwoch, 30. April 2008 02:06 To: [hidden email] Subject: FRET microscopy tips and references? I've recently been assigned a to a collaborative project that involves
trying to infer membrane protein dimerization by FRET microscopy. The proteins
being imaged have been labeled with CFP and YFP. While I understand the basics
of FRET - that when the two fluorochrome dipoles come close together the FRET
efficiency, E, goes up and this can be monitored by various ratiometric imaging
strategies - it's not clear to me how the E value can be used to
quantitatively determine dimerization, however. I'm new to this type
of imaging and was hoping someone out there could point me to a few references
that explain how FRET imaging can be used to quantitatively determine
oligimerization of proteins in general. Who was the first to do this
experimentally? Is there a way to distinguish stoichiometry (dimers vs.
trimers, etc)?
I will be performing my experiments on a multi-laser line TIRF microscope
fitted with an EMCCD camera and appropriate filter cubes. Of the FRET imaging
strategies available, which one is the best? I've read a little bit about
acceptor and donor photobleaching techniques, and these seem like the simplest
ways to determine FRET, but how accurate are these methods compared to
others?
As usual, thanks to anyone who can enlighten me (no pun
intended). John Oreopoulos, BSc, PhD Candidate University of Toronto Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging Tel: W:416-946-5022 |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi John,
what you plan to do is rather tough. FRETN is a "heavy"
compromise due to inability to accurately control concentration of donor and/or
acceptor per voxel. FLIM is a good idea, but in practical terms I wish
it would have been used on a much broader basis.
I may feel doubtful that the donor-acceptor concentration concern was
carefully and sufficiently accurately addressed in the paper mentioned by
Jens.
We live with what we have.
Dimerization versus trimerization could be discriminated, again in
theory though (it was recently published as well, but I do not recommend to go
into it unless you have >12 years to spare), let say by using split CeFP
(or venusYFP) fragments. However, in most instances, especially when
working with membrane receptors (I have experience with CXCR4, CCR5 and CD8
alpha), [Receptor-NY or -CY] constructs suffer from relatively high insolubility
in comparison to the full-length FP constructs, thus likely causing non-specific
aggregation and possibly FRET.
Virology is a kind of "taboo" these days (you won't get an RO1 grant for),
but due to a very simple and rather obvious fact (it may not be obvious to NIH
"grant judges"), the mean concentration of structural proteins tagged with donor
and acceptor per voxel (e.g. within a 130 nm HIV virus-like particle, VLP) is a
constant. I titrated donor versus acceptor from 1:9 through 9:1, the FRET
efficiency measurements by Gordon et al. 1998 showed linear
relationship. Thus, a model based on VLPs is ideal. As to the
receptors, for TIR applications artificial membrane, surface immobilized
receptors could be the best starting point. I am planning to do a
single dimer FRET experiments with CD4 and CCR5 receptors under TIR conditions
soon.
There are lots of small, often boring but important details
(venusYFP vs YFP-F64L, mCherry vs mRFP1, etc.)...
If you need these details, please contact me off-site.
Cheers,
Vitaly
NCI-Frederick,
301-846-6575
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