Bleed trhough from td-tomato using Zeiss spinning disc confocal microscope
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Julia Edgar |
Bleed trhough from td-tomato using Zeiss spinning disc confocal microscope
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Gary, George et al. Thank you for advice regarding synchronising focus between eye and camera - very helpful. Another question if you don't mind. We have a relatively weak signal (green) from EOS2, partly due to the size of the EOS2 labelled peroxisomes (approx. 50-100 nm in diameter) and a very strong signal from td-tomato in cells. By eye, using wide field epifluorescence, there is no bleed through of td-tomato into the green (EOS2) channel. But when we switch to the confocal for imaging, the bleed through is horrendous when exciting with the 488 laser. I don't know if/how to change the capture of the emitted light using this Zeiss spinning disc to get rid of the red. Is this possible and if so, how do I do it? Our Zeiss rep is on holiday and I've had no response using the generic 'support' e-mail address, hence the reason I'm posting these questions here. Many thanks in advance Best wishes Julia ............................................................................................................................. Depending on the configuration of the c-mount, you might even be able to pull it out a few mm from the stand. Best, Gary > On Oct 18, 2016, at 3:19 PM, Julia Edgar <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Many thanks George > That's what I thought I should do, but this one doesn't have adjustable eyepieces (as far as I can tell). > Best wishes > Julia > > -----Original Message----- > From: George McNamara [mailto:[hidden email]] > Sent: 18 October 2016 19:29 > To: Confocal Microscopy List; Julia Edgar > Subject: Re: Zeiss spinning disc confocal microscope > > Hi Julia, > > Best to adjust the EYEPIECES to match the camera. > > Most research microscopes have adjustable eyepieces. Typically these have a "tick mark" (or ring) to mark the eyepiece focus for 20/20 vision. Best to start with a medium to long working distance objective lens. > > Procedure: > > Get close to focus. > > Set both eyepieces to "20/20" setting. > > Use the camera to focus (if far from focus, be careful not to smash anything). > > Look in the eyepieces - should match for users with 20/20 vision. IF > NOT > - could be the user(s) do not have 20/20 vision and/or the way the camera is mounted on the microscope is "way off" (there is not much that can go wrong on a simple 1x C-mount adapter). > > Personally, I rarely used the eyepieces. Many visitors to my former microscope room at MDACC saw my (still) patent pending cover for the Leica DMI6000 microscope to keep light from entering the eyepieces and reaching the ORCA FLASH 4.2 sCMOS. I rarely removed the cover (not that I paid myself royalties to do so). > > NOTE: some objective lenses have "correction collars" for coverglass thickness or refractive index. Adjust eyepiece focus (and if needed deal with any weird camera mount issues) with a standard dry objective lens (i.e. 20x/0.8NA lens, no correction collar), then switch objective lenses, correct the correction collar to match the standard lens. > > enjoy, > > George > > p.s. hopefully the spinning disk component does not severely mess with the parfocality, or have some secret parfocality adjustment that is far from adjustment). > > >> On 10/18/2016 1:11 PM, Julia Edgar wrote: >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> Hi All >> Can anybody tell me how to adjust the camera focus so that it synchronises with the focal plane we observe by eye? >> At present we have to adjust the focal plane after switching to camera. I don't believe we should have to do this. >> Many thanks >> Best wishes >> Julia >> >> Julia M Edgar BSc(Hons), PhD, FHEA >> Institute of Infection, Immunity and Inflammation College of Medical >> Veterinary and Life Sciences University of Glasgow Sir Graeme Davies >> Building >> 120 University Place >> Glasgow >> G12 8TA >> UK >> Tel 0141 330 2082 >> https://www.facebook.com/Neuroimmunology-group-at-the-University-of-G >> l >> asgow-1741018122797881/ >> >> www.facebook.com/iiiiglasgow >> >> And >> >> Department of Neurogenetics >> Max Planck Institute for Experimental Medicine Hermann-Rein-Strasse 3 >> D-37075 Goettingen >> Germany > > -- > > > George McNamara, PhD > Houston, TX 77054 > [hidden email] > https://www.linkedin.com/in/georgemcnamara > https://works.bepress.com/gmcnamara/75/ > http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 |
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Gary Laevsky |
Re: Bleed trhough from td-tomato using Zeiss spinning disc confocal microscope
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Hi Julia, What model disc are you using?On Wed, Oct 19, 2016 at 5:20 AM, Julia Edgar <[hidden email]> wrote: ***** -- Best, Gary Laevsky, Ph.D. Director, Confocal Imaging Facility Nikon Center of Excellence Dept. of Molecular Biology Washington Rd. Princeton University Princeton, New Jersey, 08544-1014 (O) 609 258 5432 (C) 508 507 1310 |
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Jason Kirk |
Re: Bleed trhough from td-tomato using Zeiss spinning disc confocal microscope
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In reply to this post by Julia Edgar
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Julia, First I would suggest confirming which emission filter you are using as Gary states - I recommend something like a BP500-530. Most importantly - ensure you are using a bandpass filter and not a long pass filter in your configuration. A long pass filter will dramatically increase the amount of cross-excited tdTomato you detect. If you filter is labeled LP it is long pass. If you (or the user) cannot do anything to change the expression level of the tdTomato I am afraid you have run into a drawback of using a filter based system. If you are already using a bandpass filter like the one mentioned above you can try higher quality (or tighter range) bandpass filters - but that is obviously more time consuming and expensive. If you don’t to go down that road I would either change fluorophores or see if there is something you can do to reduce the expression level. Good luck! -Jason > On Oct 19, 2016, at 4:20 AM, Julia Edgar <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Dear Gary, George et al. > > Thank you for advice regarding synchronising focus between eye and camera - very helpful. > > Another question if you don't mind. > We have a relatively weak signal (green) from EOS2, partly due to the size of the EOS2 labelled peroxisomes (approx. 50-100 nm in diameter) and a very strong signal from td-tomato in cells. By eye, using wide field epifluorescence, there is no bleed through of td-tomato into the green (EOS2) channel. > But when we switch to the confocal for imaging, the bleed through is horrendous when exciting with the 488 laser. > I don't know if/how to change the capture of the emitted light using this Zeiss spinning disc to get rid of the red. Is this possible and if so, how do I do it? > > Our Zeiss rep is on holiday and I've had no response using the generic 'support' e-mail address, hence the reason I'm posting these questions here. > > Many thanks in advance > Best wishes > Julia > ............................................................................................................................. > Depending on the configuration of the c-mount, you might even be able to pull it out a few mm from the stand. > > Best, > > Gary > > > >> On Oct 18, 2016, at 3:19 PM, Julia Edgar <[hidden email]> wrote: >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> Many thanks George >> That's what I thought I should do, but this one doesn't have adjustable eyepieces (as far as I can tell). >> Best wishes >> Julia >> >> -----Original Message----- >> From: George McNamara [mailto:[hidden email]] >> Sent: 18 October 2016 19:29 >> To: Confocal Microscopy List; Julia Edgar >> Subject: Re: Zeiss spinning disc confocal microscope >> >> Hi Julia, >> >> Best to adjust the EYEPIECES to match the camera. >> >> Most research microscopes have adjustable eyepieces. Typically these have a "tick mark" (or ring) to mark the eyepiece focus for 20/20 vision. Best to start with a medium to long working distance objective lens. >> >> Procedure: >> >> Get close to focus. >> >> Set both eyepieces to "20/20" setting. >> >> Use the camera to focus (if far from focus, be careful not to smash anything). >> >> Look in the eyepieces - should match for users with 20/20 vision. IF >> NOT >> - could be the user(s) do not have 20/20 vision and/or the way the camera is mounted on the microscope is "way off" (there is not much that can go wrong on a simple 1x C-mount adapter). >> >> Personally, I rarely used the eyepieces. Many visitors to my former microscope room at MDACC saw my (still) patent pending cover for the Leica DMI6000 microscope to keep light from entering the eyepieces and reaching the ORCA FLASH 4.2 sCMOS. I rarely removed the cover (not that I paid myself royalties to do so). >> >> NOTE: some objective lenses have "correction collars" for coverglass thickness or refractive index. Adjust eyepiece focus (and if needed deal with any weird camera mount issues) with a standard dry objective lens (i.e. 20x/0.8NA lens, no correction collar), then switch objective lenses, correct the correction collar to match the standard lens. >> >> enjoy, >> >> George >> >> p.s. hopefully the spinning disk component does not severely mess with the parfocality, or have some secret parfocality adjustment that is far from adjustment). >> >> >>> On 10/18/2016 1:11 PM, Julia Edgar wrote: >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> Post images on http://www.imgur.com and include the link in your posting. >>> ***** >>> >>> Hi All >>> Can anybody tell me how to adjust the camera focus so that it synchronises with the focal plane we observe by eye? >>> At present we have to adjust the focal plane after switching to camera. I don't believe we should have to do this. >>> Many thanks >>> Best wishes >>> Julia >>> >>> Julia M Edgar BSc(Hons), PhD, FHEA >>> Institute of Infection, Immunity and Inflammation College of Medical >>> Veterinary and Life Sciences University of Glasgow Sir Graeme Davies >>> Building >>> 120 University Place >>> Glasgow >>> G12 8TA >>> UK >>> Tel 0141 330 2082 >>> https://www.facebook.com/Neuroimmunology-group-at-the-University-of-G >>> l >>> asgow-1741018122797881/ >>> >>> www.facebook.com/iiiiglasgow >>> >>> And >>> >>> Department of Neurogenetics >>> Max Planck Institute for Experimental Medicine Hermann-Rein-Strasse 3 >>> D-37075 Goettingen >>> Germany >> >> -- >> >> >> George McNamara, PhD >> Houston, TX 77054 >> [hidden email] >> https://www.linkedin.com/in/georgemcnamara >> https://works.bepress.com/gmcnamara/75/ >> http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 |
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Claire Brown |
Re: Bleed trhough from td-tomato using Zeiss spinning disc confocal microscope
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In reply to this post by Julia Edgar
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Julia, I would suggest this is not bleedthrough at all but direct excitation of the tdTomato by the 488 light. If you look at the tdTomato excitation spectra it is pretty efficient at absorbing 488 nm light. A couple of suggestions: 1) Minimize the intensity of the 488 nm light as much as possible and maximize your exposure time. I know this will be at the expense of speed. This combined with narrow band pass could do the trick. It is very counter intuitive but when you have a dim signal the best thing to do is put the excitation light down. This minimizes photobleaching and you can get much more signal out of the molecules you do have around. 2) If you have access to lower wavelength lasers like the 458 nm line from the Argon try that. You could even try the 405 nm laser for EGFP. I know it is not typical but it might give you enough excitation and should not excite the tdTomato at all. 3) With whichever components you use you can also do a cross-talk correction. This would be direct excitation cross-talk and you could have some emission cross-talk from the EGFP as well. You need a sample with EGFP alone - image it in both the EGFP and tdTomato channels and calculate the % emission cross-talk. Then take a td-tomato alone sample and image it in the EGFP and tdTomato channels with the EGFP excitation and measure your direct excitation cross-talk. It is crucial to use the exact same settings as you use for two colour imaging for these controls. I'm happy to discuss further offline if you like. 4) Lastly, choose a different red fluorescent protein that does not excite well at 488 nm. mCherry or TagRFP should be better. Good luck! Claire |
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Sylvie Le Guyader |
Re: Bleed trhough from td-tomato using Zeiss spinning disc confocal microscope
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In reply to this post by Julia Edgar
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Hi Julia On our
website, under teaching material, we will find a video that will guide you through all the steps to avoid bleed through when designing your sample. In the video we refer to a quiz table that our students need to fill in. This document is just above the
videos (Spinning_disk_excitation_and_emission). You can make your own using the specs of your excitation source and emission filters. Hope that helps :) Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Hälsovägen 7, Novum, G lift, floor 6 14157 Huddinge Sweden mobile: +46 (0) 73 733 5008 office: +46 (0) 08-524 811 72 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julia Edgar Sent: den 19 oktober 2016 11:21 To: [hidden email] Subject: Bleed trhough from td-tomato using Zeiss spinning disc confocal microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Gary, George et al. Thank you for advice regarding synchronising focus between eye and camera - very helpful. Another question if you don't mind. We have a relatively weak signal (green) from EOS2, partly due to the size of the EOS2 labelled peroxisomes (approx. 50-100 nm in diameter) and a very strong signal from td-tomato in cells. By eye, using wide field epifluorescence, there
is no bleed through of td-tomato into the green (EOS2) channel. But when we switch to the confocal for imaging, the bleed through is horrendous when exciting with the 488 laser. I don't know if/how to change the capture of the emitted light using this Zeiss spinning disc to get rid of the red. Is this possible and if so, how do I do it? Our Zeiss rep is on holiday and I've had no response using the generic 'support' e-mail address, hence the reason I'm posting these questions here. Many thanks in advance Best wishes Julia |
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Julia Edgar |
Re: Bleed trhough from td-tomato using Zeiss spinning disc confocal microscope
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*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****
Dear All Thanks so much for your very helpful contributions. Very much appreciated. Best wishes Julia From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Sylvie Le Guyader ***** To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on
http://www.imgur.com and include the link in your posting. *****
Hi Julia On our
website, under teaching material, we will find a video that will guide you through all the steps to avoid bleed through when designing your sample. In the video we refer to a quiz table that our
students need to fill in. This document is just above the videos (Spinning_disk_excitation_and_emission). You can make your own using the specs of your excitation source and emission filters. Hope that helps :) Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Hälsovägen 7,
Novum, G lift, floor 6 14157 Huddinge Sweden mobile: +46 (0) 73 733 5008 office: +46 (0) 08-524 811 72 -----Original Message----- From: Confocal Microscopy List [[hidden email]] On Behalf Of Julia Edgar Sent: den 19 oktober 2016 11:21 To:
[hidden email] Subject: Bleed trhough from td-tomato using Zeiss spinning disc confocal microscope ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on
http://www.imgur.com and include the link in your posting. ***** Dear Gary, George et al. Thank you for advice regarding synchronising focus between eye and camera - very helpful. Another question if you don't mind. We have a relatively weak signal (green) from EOS2, partly due to the size of the EOS2 labelled peroxisomes (approx. 50-100 nm in diameter) and a very strong signal from td-tomato
in cells. By eye, using wide field epifluorescence, there is no bleed through of td-tomato into the green (EOS2) channel. But when we switch to the confocal for imaging, the bleed through is horrendous when exciting with the 488 laser. I don't know if/how to change the capture of the emitted light using this Zeiss spinning disc to get rid of the red. Is this possible and if so, how do I do it? Our Zeiss rep is on holiday and I've had no response using the generic 'support' e-mail address, hence the reason I'm posting these questions here. Many thanks in advance Best wishes Julia |
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