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Hi,
I'm having trouble locating a commercial vendor that sells Congo red dye. Is there another name for this dye and can anyone direct me to a site that sells Congo red? And can someone out there show me an absorption and emission spectrum of this dye? Thanks! 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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We got ours from Fluka, cat no. 60910.
Cheers, Howard On Dec 6, 2007, at 11:24 AM, John Oreopoulos wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal R. Howard Berg, Ph.D. Director, Integrated Microscopy Facility Danforth Plant Science Center 975 N. Warson Rd. St. Louis, MO 63132 ph 314-587-1261 fx 314-587-1361 cell 314-378-2409 visit this educational resource: http://www.danforthcenter.org/Cells/ |
In reply to this post by John Oreopoulos
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John,
At 12:24 PM -0500 12/6/07, John Oreopoulos wrote:
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, I'm having trouble locating a commercial vendor that sells Congo red dye. Is there another name for this dye and can anyone direct me to a site that sells Congo red? And can someone out there show me an absorption and emission spectrum of this dye? Thanks! 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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In reply to this post by John Oreopoulos
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-
here are some references: last reference lists the product as purchased from SIGMA (C-6277) You will get more info if you Google "congo red" -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On Dec 6, 2007, at 9:24 AM, John Oreopoulos wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, |
James Pawley |
In reply to this post by Martin Wessendorf
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Hi all,
While there are definitely dye-choice/bleaching/cytotoxicity/ problems with STED, I don't think that it is quite as bad as Martin calculates. This is because the various damage mechanisms are all proportional to the time that the dye is in the singlet-excited state. And, as the STEp pulse follows the excitation pulse by a few ps, each molecule that is quenched by this pulse is only excited for a few ps, not for the few ns that characterize its normal fluorescence lifetime. Of the other hand, most STED is done with pulsed beams and, as this means that dye molecules are neither excited or being stimulated at least 90% of the time, the signal level is lower and one must operate 10x closer to the singlet-state saturation. This will probably lead to more "1+1 photon" damage and enhanced bleaching. In addition, as has been pointed out before, unless the dyes molecules are concentrated into very small nm-scale structures (i.e., a ribosome or a centriole), having a smaller effective excitation volume means that fewer dyes molecules will be located in each volume and this will either reduce signal or require increased pixel-dwell time. STED is a very clever technique, however, I think that over the long run, its widespread application will be determined in competition with correlated LM/EM microscopy. One really has to compare the utility of one STED vs. about 4-5 standard confocals. Happy Holidays! Jim P. On Dec 6, 2007, at 10:51 AM, Martin Wessendorf wrote: Search the CONFOCAL archive at Prof. James B. Pawley, Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ "If it ain't diffraction, it must be statistics." Anon. |
Mark Cannell |
In reply to this post by Martin Wessendorf
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Martin Perhaps you could expand on this, I'd have thought that if the fluorophore is depleted by stimulated emission with I/Isat > 1 it is in the excited state for very much less time. Cheers Mark On 7/12/2007, at 5:51 AM, Martin Wessendorf wrote: > > > Interesting question. Someone pointed out to me off-list that with > STED, the fluorophore gets taken up to the excited state many more > times (--presumably 25x for a 5x increase in resolution) than with > conventional multiphoton microscopy for each photon of actual > fluorescence. However, for each time it's in the excited state, it > has the same probability of bad photochemistry occuring--e.g., > transition to the triplet state and/or phototoxicity. In this > example, you'd expect 25x more phototoxicity with STED than with > multiphoton in order to obtain the same image. Thus there may be > inherent limitations with STED with regards to live-cell imaging. > > |
Martin Wessendorf |
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hey, Mark-- Mark Cannell wrote: > Perhaps you could expand on this, I'd have thought that if the > fluorophore is depleted by stimulated emission with I/Isat > 1 it is in > the excited state for very much less time. I think that you and Jim Pawley make the same point--correctly, as nearly as my feeble quantum mechanics can apprehend! As Jim and others have pointed out, the dwell time per unit area will be increased, and that will tend to increase both photobleaching and phototoxicity. But no, it appears that the increased cycling of the fluorophore shouldn't be much of an issue. Martin -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 E-mail: martinw[at]med.umn.edu |
In reply to this post by John Oreopoulos
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Hi John,
http://www.usca.edu/chemistry/spectra/ has the UV-VIS (absorption spectrum) for Congo Red and the other dyes in Jack Goldsmith's web site is in the no longer supported Thermo Galactic ".p7b" file format. All Jack's spectra data are in our standard format in one of our PubSpectra Excel files in http://home.earthlink.net/~gfpology/McNamara_Boswell_Spectra_ Dyes_FPs .zip (the one with Goldsmith in the name). Absorption spectra page 220 of Green 1990 Sigma-Aldrich Handbook of Dyes and Indicators, on page 490 of Gurr 1971 Synthetic Dyes in Biology and Medicine, and W.E. Klunk 1999 Analytical Biochemistry 266: 66-76. Emission spectrum is in fig 2 of A. Elhaddaoui 1995 Biospectroscopy 1: 351-356. Spectra may differ in solution and bound to amyloid, etc. George p.s. another link for histology stains is http://stainsfile.info/StainsFile/dyes/22120.htm At 12:24 PM 12/6/2007, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, George McNamara, Ph.D. University of Miami, Miller School of Medicine Image Core Miami, FL 33010 [hidden email] [hidden email] 305-243-8436 office http://home.earthlink.net/~pubspectra/ http://home.earthlink.net/~geomcnamara/ http://www.sylvester.org/health_pro/shared_resources/index.asp (see Analytical Imaging Core Facility) |
In reply to this post by Michael Weber-4
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, from the department of Stefan Hell this problem has been tackled from many point of view. First of all, the repeteation rate can be optimized in order to minimize photobleaching (see D-Rex [PMID: 17179937] illumination and STED [I think PMID: 17307826 is the correct one]) and a generalization of STED (RESOLFT - [PMID: 16314572]) was proposed that makes use of lower intensities at the sample. On regard of photobleaching alone, they are trying to develop better fluorophores. The last example is PMID: 18058955. STORM/PALM/PALMIRA may become faster then currently is, but these techniques will be always limited by the necessity of sequential imaging of each single fluorophore. I am looking forward for fluorophores optimized for these kind of techniques and I am quite sure that other optimization/innovations are possible... Cheers, -- Dr. Alessandro Esposito Laser Analytics Group - Department of Chemical Engineering University Cambridge E.mail: [hidden email] CV: http://home.quantitative-microscopy.org Web: http://www.wikiscope.org |
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