Reverse Bleed-through revisted
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Hello Group,
In January there was a post and comments regarding Cy3 photobleaching of DAPI and I have re-read them trying to come up with an explanation for something I was shown today on my system. A user has fixed cells stained with DAPI, stably expressing GFP, and targets stained indirectly with Alexa 555 and Alexa 647. We are seeing what seems to be the Alexa 555 signal in the GFP channel. We are illuminating with an EXFO Hg lamp, and using the Semrock Quad-band Sedat filter set. The excitation, emission and dichroic mirror specs from the Semrock website are provided below. A small amount of Alexa 555 is excited by the GFP excitation indicated below, but the cut-off on the GFP EM should preclude any Alexa 555 EM getting through. I am not sure if I am interpreting the Mol Probes Spectraviewer correctly and therefore I am asking the group for thoughts. We are obtaining what appears to be Alexa 555 signal in the GFP channel at very long exposure times (400ms) and high gain (>3800 on a scale max'ing at 4095) on a Photometrics Cascade II EM-CCD, imaging in widefield. Apologies if this seems to be an amateurish question, but I can't seem to figure it out. Thanks very much all. Cheers Farid
Farid Jalali MSc Senior Research Technician- Lab Manager Applied Molecular Oncology/ Princess Margaret Hospital STTARR Innovation Facility/ Radiation Medicine Program Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Microscopy Suit |
 
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Aryeh Weiss |
Re: Reverse Bleed-through revisted
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Farid Jalali wrote: > We are seeing what seems to be the Alexa 555 > signal in the GFP channel. We are illuminating with an EXFO Hg lamp, > and using the Semrock Quad-band Sedat filter set. The excitation, > emission and dichroic mirror specs from the Semrock website are provided > below. A small amount of Alexa 555 is excited by the GFP excitation > indicated below, but the cut-off on the GFP EM should preclude any Alexa > 555 EM getting through. I am not sure if I am interpreting the Mol > Probes Spectraviewer correctly and therefore I am asking the group for > thoughts. We are obtaining what appears to be Alexa 555 signal in the > GFP channel at very long exposure times (400ms) and high gain (>3800 on > a scale max'ing at 4095) on a Photometrics Cascade II EM-CCD, imaging in > widefield. When you excite a longer wavelength fluorophore with a short wavelength, you can get emission from the short wavelength "tail" of the emission spectrum. The various spectrum viewer show the spectra on a linear scale, so it looks like there is no emission, but that is not necessarily the case. In many cases, the spectral data are not provided so far from the peaks, but again, that does not mean that there is absolutely nothing. You are detecting at very high gain, and perhaps you have strong staining with the alexa555. If you want to test this, you can run spectra of alexa555 in a high quality spectrofluorometer, and acquire the emission spectrum with 485nm excitation. --aryeh -- Aryeh Weiss School of Engineering Bar Ilan University Ramat Gan 52900 Israel Ph: 972-3-5317638 FAX: 972-3-7384050 |
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Glen MacDonald-2 |
Re: Reverse Bleed-through revisted
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In reply to this post by Farid Jalali
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Farid, As Aryeh says, check the spectra. You can't rely on the stated maxima to judge bleedthrough or cross talk. If you check EGFP and Alexa555 on a spectra viewer, like at Molecular Probes, you will see a small overlap from the blue side of the A555 excitation curve and your exciter filter. Small, but enough to generate enough photons to be a problem when sufficiently amplified by long exposures with a cranked up EMCCD. You are amplifying everything, regardless of the source. I'd suggest switching to Alexa568, unless you can either cut exposure time or gain to minimize the problem, or move to a blue- shifted excitation for the GFP. Regards, Glen On Jul 31, 2008, at 7:47 PM, Farid Jalali wrote: > Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Hello Group, > In January there was a post and comments regarding Cy3 > photobleaching of DAPI and I have re-read them trying to come up > with an explanation for something I was shown today on my system. A > user has fixed cells stained with DAPI, stably expressing GFP, and > targets stained indirectly with Alexa 555 and Alexa 647. We are > seeing what seems to be the Alexa 555 signal in the GFP channel. We > are illuminating with an EXFO Hg lamp, and using the Semrock Quad- > band Sedat filter set. The excitation, emission and dichroic mirror > specs from the Semrock website are provided below. A small amount of > Alexa 555 is excited by the GFP excitation indicated below, but the > cut-off on the GFP EM should preclude any Alexa 555 EM getting > through. I am not sure if I am interpreting the Mol Probes > Spectraviewer correctly and therefore I am asking the group for > thoughts. We are obtaining what appears to be Alexa 555 signal in > the GFP channel at very long exposure times (400ms) and high gain > (>3800 on a scale max'ing at 4095) on a Photometrics Cascade II EM- > CCD, imaging in widefield. > Apologies if this seems to be an amateurish question, but I can't > seem to figure it out. > > Thanks very much all. > Cheers > Farid > > Filter > Average Transmission > Center Wavelengths > Bandwidths * > DAPI EX > > 85% > 387 nm > 11 nm > GFP EX > > 90% > 485 nm > 20 nm > Alexa555 EX > > 90% > 560 nm > 25 nm > Alexa647 EX > > 90% > 650 nm > 13 nm > DAPI EM > > 90% > 440 nm > 40 nm > GFP EM > > 90% > 525 nm > 30 nm > Alexa555 EM > > 90% > 607 nm > 36 nm > Alexa647 EM > > 90% > > 684 nm > 24 nm > > Average Reflection > Reflection Bands > Transmission Bands > Dichroic > > 95% > > 95% > > 95% > > 95% > 381 - 392 nm > 475 - 495 nm > 547 - 572 nm > 643 - 656 nm > 420 - 460 nm > 510 - 531 nm > 589 - 623 nm > 677 - 722 nm > -- > Farid Jalali MSc > Senior Research Technician- Lab Manager > Applied Molecular Oncology/ Princess Margaret Hospital > STTARR Innovation Facility/ Radiation Medicine Program > Toronto, Canada > 416-946-4501 X4351 (Princess Margaret Hospital) > 416-581-7754 STTARR at MaRS Building > 416-581-7791 STTARR Microscopy Suit 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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Greg Martin-8 |
Re: Reverse Bleed-through revisted
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hey Folks -- This has been a common problem with Cy3 as well, even without particularly sensitive cameras. I've learned and teach that regardless of what the spectra say, it's always best to actually do the experiment and see what you get with a single label viewed in the "wrong" channel using exposure and scaling appropriate to that channel. Be peace! Greg. Greg Martin Keck Microscopy Facility University of Washington Box 357290 Seattle, WA 98195-7290 206-685-8784 (office) 425-344-2632 (cell) www.depts.washington.edu/keck ----- Original Message ----- From: "Glen MacDonald" <[hidden email]> To: <[hidden email]> Sent: Friday, August 01, 2008 9:10 AM Subject: Re: Reverse Bleed-through revisted > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Farid, > As Aryeh says, check the spectra. You can't rely on the stated maxima to > judge bleedthrough or cross talk. If you check EGFP and Alexa555 on a > spectra viewer, like at Molecular Probes, you will see a small overlap > from the blue side of the A555 excitation curve and your exciter filter. > Small, but enough to generate enough photons to be a problem when > sufficiently amplified by long exposures with a cranked up EMCCD. You are > amplifying everything, regardless of the source. > > I'd suggest switching to Alexa568, unless you can either cut exposure > time or gain to minimize the problem, or move to a blue- shifted > excitation for the GFP. > > Regards, > Glen > > > On Jul 31, 2008, at 7:47 PM, Farid Jalali wrote: > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> Hello Group, >> In January there was a post and comments regarding Cy3 photobleaching of >> DAPI and I have re-read them trying to come up with an explanation for >> something I was shown today on my system. A user has fixed cells stained >> with DAPI, stably expressing GFP, and targets stained indirectly with >> Alexa 555 and Alexa 647. We are seeing what seems to be the Alexa 555 >> signal in the GFP channel. We are illuminating with an EXFO Hg lamp, >> and using the Semrock Quad- band Sedat filter set. The excitation, >> emission and dichroic mirror specs from the Semrock website are provided >> below. A small amount of Alexa 555 is excited by the GFP excitation >> indicated below, but the cut-off on the GFP EM should preclude any Alexa >> 555 EM getting through. I am not sure if I am interpreting the Mol >> Probes Spectraviewer correctly and therefore I am asking the group for >> thoughts. We are obtaining what appears to be Alexa 555 signal in the >> GFP channel at very long exposure times (400ms) and high gain (>3800 on >> a scale max'ing at 4095) on a Photometrics Cascade II EM- CCD, imaging in >> widefield. >> Apologies if this seems to be an amateurish question, but I can't seem >> to figure it out. >> >> Thanks very much all. >> Cheers >> Farid >> >> Filter >> Average Transmission >> Center Wavelengths >> Bandwidths * >> DAPI EX >> > 85% >> 387 nm >> 11 nm >> GFP EX >> > 90% >> 485 nm >> 20 nm >> Alexa555 EX >> > 90% >> 560 nm >> 25 nm >> Alexa647 EX >> > 90% >> 650 nm >> 13 nm >> DAPI EM >> > 90% >> 440 nm >> 40 nm >> GFP EM >> > 90% >> 525 nm >> 30 nm >> Alexa555 EM >> > 90% >> 607 nm >> 36 nm >> Alexa647 EM >> > 90% >> >> 684 nm >> 24 nm >> >> Average Reflection >> Reflection Bands >> Transmission Bands >> Dichroic >> > 95% >> > 95% >> > 95% >> > 95% >> 381 - 392 nm >> 475 - 495 nm >> 547 - 572 nm >> 643 - 656 nm >> 420 - 460 nm >> 510 - 531 nm >> 589 - 623 nm >> 677 - 722 nm >> -- >> Farid Jalali MSc >> Senior Research Technician- Lab Manager >> Applied Molecular Oncology/ Princess Margaret Hospital >> STTARR Innovation Facility/ Radiation Medicine Program >> Toronto, Canada >> 416-946-4501 X4351 (Princess Margaret Hospital) >> 416-581-7754 STTARR at MaRS Building >> 416-581-7791 STTARR Microscopy Suit > > > > 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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Knecht, David |
Re: Reverse Bleed-through revisted
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In reply to this post by Farid Jalali
Search the CONFOCAL archive at
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It looks to me like you are getting the early tail of the emission of Alexa 555 through your GFP filer. You have a 525/30, which cuts off around 550, but the Alexa emission starts up around 538nm.. You could do as others suggested, but if you already have samples stained, you could switch to a narrower GFP filter, such as the Chroma 41020 which cuts off around 520 and should eliminate the problem. Dave
On Jul 31, 2008, at 10:47 PM, Farid Jalali wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Farid Jalali |
Re: Reverse Bleed-through revisted
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hello Group,
Thanks very much to you all for the helpful thoughts and comments. Much appreciated. This user was un-necessarily using a long exposure time and gain to pick up very very weak GFP signal. This bleedthrough is not something that I have ever seen or documented on my system. Good thing to be aware of as well a point to reinforce to users. Thanks again group. Cheers Farid On Sat, Aug 2, 2008 at 1:54 PM, David Knecht ATT <[hidden email]> wrote:
-- Farid Jalali MSc Senior Research Technician- Lab Manager Applied Molecular Oncology/ Princess Margaret Hospital STTARR Innovation Facility/ Radiation Medicine Program Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Microscopy Suite |
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