co-planar localization
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello all; Interesting volume localization and quantitation question for you. One of my users wants to show that her marker moves to the plasma membrane after stimulation. We have used Alexa 568 WGA on her cells to label the membrane. Her channel protein is eGFP-labeled. Both signals are not continuous, but are punctate. After stimulation, the signal appears to move to the membrane, but the WGA and her eGFP do not overlap, i.e., the signals are found in exclusive patches, as shown by LSM confocal, 60x, zoom x2, 1.4NA in Z-stacks. I have thresholded the WGA signal in MetaMorph (with a variety of values), made a binary mask, and overlaid this on the GFP signal. What we see is the suggestion that the channel protein is at the membrane in the same plane as the WGA, but there is minimal overlap (as determined by line-scans). How to quantify this? She likes my reference to the patches on a soccer ball...they are obviously in the same spherical plane, but do not overlap. Would volume rendering and modeling help? Thanks. Kathy Kathryn Spencer, Ph.D. The Scripps Research Institute ICND 210 10550 N. Torrey Pines Road La Jolla, CA 92037 (858) 784-8437 [hidden email] |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Kathy, How about trying a Di dye? They are more ubiquitous in the membrane and you could get them in somewhat similar (DiI ex/em at 549/565)spectra. No Commercial Interest Best, Gary Laevsky, Ph.D. Imaging Application Specialist Andor Technology discover new ways of seeing [hidden email] Cell (774) 291 - 9992 Office (860) 290 - 9211 x219 Fax (860) 290 - 9566 Web: www.andor.com -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kathryn Spencer Sent: Wednesday, August 06, 2008 4:44 PM To: [hidden email] Subject: co-planar localization Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello all; Interesting volume localization and quantitation question for you. One of my users wants to show that her marker moves to the plasma membrane after stimulation. We have used Alexa 568 WGA on her cells to label the membrane. Her channel protein is eGFP-labeled. Both signals are not continuous, but are punctate. After stimulation, the signal appears to move to the membrane, but the WGA and her eGFP do not overlap, i.e., the signals are found in exclusive patches, as shown by LSM confocal, 60x, zoom x2, 1.4NA in Z-stacks. I have thresholded the WGA signal in MetaMorph (with a variety of values), made a binary mask, and overlaid this on the GFP signal. What we see is the suggestion that the channel protein is at the membrane in the same plane as the WGA, but there is minimal overlap (as determined by line-scans). How to quantify this? She likes my reference to the patches on a soccer ball...they are obviously in the same spherical plane, but do not overlap. Would volume rendering and modeling help? Thanks. Kathy Kathryn Spencer, Ph.D. The Scripps Research Institute ICND 210 10550 N. Torrey Pines Road La Jolla, CA 92037 (858) 784-8437 [hidden email] |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Results were UGLY...WGA was nice and specific, but too punctate. What's new out your way? Are you liking Andor? Scripps is still plugging along. We have people moving in and out of ICND, but all imaging types. We have someone coming in a few weeks with her own multi-photon...finally I get to play! Kathy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gary Laevsky Sent: Wednesday, August 06, 2008 4:23 PM To: [hidden email] Subject: Re: co-planar localization Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Kathy, How about trying a Di dye? They are more ubiquitous in the membrane and you could get them in somewhat similar (DiI ex/em at 549/565)spectra. No Commercial Interest Best, Gary Laevsky, Ph.D. Imaging Application Specialist Andor Technology discover new ways of seeing [hidden email] Cell (774) 291 - 9992 Office (860) 290 - 9211 x219 Fax (860) 290 - 9566 Web: www.andor.com -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kathryn Spencer Sent: Wednesday, August 06, 2008 4:44 PM To: [hidden email] Subject: co-planar localization Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello all; Interesting volume localization and quantitation question for you. One of my users wants to show that her marker moves to the plasma membrane after stimulation. We have used Alexa 568 WGA on her cells to label the membrane. Her channel protein is eGFP-labeled. Both signals are not continuous, but are punctate. After stimulation, the signal appears to move to the membrane, but the WGA and her eGFP do not overlap, i.e., the signals are found in exclusive patches, as shown by LSM confocal, 60x, zoom x2, 1.4NA in Z-stacks. I have thresholded the WGA signal in MetaMorph (with a variety of values), made a binary mask, and overlaid this on the GFP signal. What we see is the suggestion that the channel protein is at the membrane in the same plane as the WGA, but there is minimal overlap (as determined by line-scans). How to quantify this? She likes my reference to the patches on a soccer ball...they are obviously in the same spherical plane, but do not overlap. Would volume rendering and modeling help? Thanks. Kathy Kathryn Spencer, Ph.D. The Scripps Research Institute ICND 210 10550 N. Torrey Pines Road La Jolla, CA 92037 (858) 784-8437 [hidden email] |
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In reply to this post by kspencer007
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, In my opinion there is not really a problem here, or at least no more of a problem than there would be if the patches would be overlapping. The fact that you see both types of patches in the same focal plane means obviously that they are in the same plane within the microscope's resolution. If the patches would be visually overlapping you would still have the same uncertainty with regard to the z-position of both. It just looks a bit nicer, that's all. Colocalisation based on the coincidence of colors in still images is always limited by the spatial resolution. Best regards, Kevin > -----Oorspronkelijk bericht----- > Van: Confocal Microscopy List [mailto:[hidden email]] > Namens Kathryn Spencer > Verzonden: woensdag 6 augustus 2008 22:44 > Aan: [hidden email] > Onderwerp: co-planar localization > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello all; > Interesting volume localization and quantitation question for > you. > One of my users wants to show that her marker moves to the > plasma membrane after stimulation. We have used Alexa 568 WGA on her > cells to label the membrane. Her channel protein is eGFP-labeled. Both > signals are not continuous, but are punctate. After stimulation, the > signal appears to move to the membrane, but the WGA and her eGFP do not > overlap, i.e., the signals are found in exclusive patches, as shown by > LSM confocal, 60x, zoom x2, 1.4NA in Z-stacks. I have thresholded the > WGA signal in MetaMorph (with a variety of values), made a binary mask, > and overlaid this on the GFP signal. What we see is the suggestion that > the channel protein is at the membrane in the same plane as the WGA, > but > there is minimal overlap (as determined by line-scans). How to quantify > this? She likes my reference to the patches on a soccer ball...they are > obviously in the same spherical plane, but do not overlap. Would volume > rendering and modeling help? > Thanks. > Kathy > > > > Kathryn Spencer, Ph.D. > The Scripps Research Institute > ICND 210 > 10550 N. Torrey Pines Road > La Jolla, CA 92037 > (858) 784-8437 > [hidden email] |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have a Leica DMRE upright microscope with our Leica TCS SP2 confocal system and are interested in sourcing a Dark Field condenser bottom for it. This part is no longer able to be supplied by Leica and I was wondering/hoping that someone out there may have a spare one, or one they longer need or want that they are willing to sell for a reasonable price. The old Leica part number is 11 505 078. Many thanks in advance. Kind Regards, Grace Grace Chojnowski Flow Cytometry and Confocal Microscopy Laboratory Manager Queensland Institute of Medical Research Brisbane, QLD, AUSTRALIA Email: [hidden email] Telephone 61-7-3362 0314. Mob: 0414 956 752 FAX 61-7-3362 0107 |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal you might want to look into the Cytoviva system. (http://www.cytoviva.com/main.htm) .. probably the ultimate darkfield condenser.. Bill Miller At 05:28 PM 8/7/2008 +1000, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >We have a Leica DMRE upright microscope with our Leica TCS SP2 confocal >system and are interested in sourcing a Dark Field condenser bottom for >it. >This part is no longer able to be supplied by Leica and I was >wondering/hoping that someone out there may have a spare one, or one >they longer need or want that they are willing to sell for a reasonable >price. The old Leica part number is 11 505 078. > >Many thanks in advance. > >Kind Regards, > >Grace > > Grace Chojnowski > Flow Cytometry and Confocal Microscopy Laboratory Manager > Queensland Institute of Medical Research > Brisbane, QLD, AUSTRALIA > Email: [hidden email] > Telephone 61-7-3362 0314. Mob: 0414 956 752 > FAX 61-7-3362 0107 |
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In reply to this post by Kevin Braeckmans
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Sounds to me like a TIRF microscope might help... Cheers Mark Cannell Kevin Braeckmans wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi, > > In my opinion there is not really a problem here, or at least no more of a > problem than there would be if the patches would be overlapping. The fact > that you see both types of patches in the same focal plane means obviously > that they are in the same plane within the microscope's resolution. If the > patches would be visually overlapping you would still have the same > uncertainty with regard to the z-position of both. It just looks a bit > nicer, that's all. Colocalisation based on the coincidence of colors in > still images is always limited by the spatial resolution. > > Best regards, > > Kevin > > > >> -----Oorspronkelijk bericht----- >> Van: Confocal Microscopy List [mailto:[hidden email]] >> Namens Kathryn Spencer >> Verzonden: woensdag 6 augustus 2008 22:44 >> Aan: [hidden email] >> Onderwerp: co-planar localization >> >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hello all; >> Interesting volume localization and quantitation question for >> you. >> One of my users wants to show that her marker moves to the >> plasma membrane after stimulation. We have used Alexa 568 WGA on her >> cells to label the membrane. Her channel protein is eGFP-labeled. Both >> signals are not continuous, but are punctate. After stimulation, the >> signal appears to move to the membrane, but the WGA and her eGFP do not >> overlap, i.e., the signals are found in exclusive patches, as shown by >> LSM confocal, 60x, zoom x2, 1.4NA in Z-stacks. I have thresholded the >> WGA signal in MetaMorph (with a variety of values), made a binary mask, >> and overlaid this on the GFP signal. What we see is the suggestion that >> the channel protein is at the membrane in the same plane as the WGA, >> but >> there is minimal overlap (as determined by line-scans). How to quantify >> this? She likes my reference to the patches on a soccer ball...they are >> obviously in the same spherical plane, but do not overlap. Would volume >> rendering and modeling help? >> Thanks. >> Kathy >> >> >> >> Kathryn Spencer, Ph.D. >> The Scripps Research Institute >> ICND 210 >> 10550 N. Torrey Pines Road >> La Jolla, CA 92037 >> (858) 784-8437 >> [hidden email] >> |
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In reply to this post by Grace Chojnowski
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Grace, Bill Miller is right it works extremely well. Contact SciTech as they are the local distributor for Cytoviva. Cheers Steve -----Original Message----- From: Grace Chojnowski [mailto:[hidden email]] Sent: Thursday, 7 August 2008 5:28 PM To: [hidden email] Subject: Darkfield Condenser Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have a Leica DMRE upright microscope with our Leica TCS SP2 confocal system and are interested in sourcing a Dark Field condenser bottom for it. This part is no longer able to be supplied by Leica and I was wondering/hoping that someone out there may have a spare one, or one they longer need or want that they are willing to sell for a reasonable price. The old Leica part number is 11 505 078. Many thanks in advance. Kind Regards, Grace Grace Chojnowski Flow Cytometry and Confocal Microscopy Laboratory Manager Queensland Institute of Medical Research Brisbane, QLD, AUSTRALIA Email: [hidden email] Telephone 61-7-3362 0314. Mob: 0414 956 752 FAX 61-7-3362 0107 |
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In reply to this post by kspencer007
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal What is that you want to quantify, Kathy? If it is the spatial relations between the labeled structures in the plane of the membrane, there is a range of spatial statistics that let you decide if they are really associated or not (eg as compared with a uniform or random distribution). Doing this in 3D can be a bit of a trick, but there are several approaches that are not too difficult, as long as you can get some quantitative data out of your image sets (typically x-y-z coordinates and grey-scale values of the features of interest.) In my experience, this proviso is surprisingly difficult to extract easily from imaging programs that must have the data embedded within them. I haven't looked at MetaMorph for a while, so I can't offer any clues there. May be someone else can? If this approach is what you are after, let me know, and I can give you some references. IAN On Thursday, August 7, 2008, at 06:14 AM, Kathryn Spencer wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello all; > Interesting volume localization and quantitation question for > you. > One of my users wants to show that her marker moves to the > plasma membrane after stimulation. We have used Alexa 568 WGA on her > cells to label the membrane. Her channel protein is eGFP-labeled. Both > signals are not continuous, but are punctate. After stimulation, the > signal appears to move to the membrane, but the WGA and her eGFP do not > overlap, i.e., the signals are found in exclusive patches, as shown by > LSM confocal, 60x, zoom x2, 1.4NA in Z-stacks. I have thresholded the > WGA signal in MetaMorph (with a variety of values), made a binary mask, > and overlaid this on the GFP signal. What we see is the suggestion that > the channel protein is at the membrane in the same plane as the WGA, > but > there is minimal overlap (as determined by line-scans). How to quantify > this? She likes my reference to the patches on a soccer ball...they are > obviously in the same spherical plane, but do not overlap. Would volume > rendering and modeling help? > Thanks. > Kathy > > > > Kathryn Spencer, Ph.D. > The Scripps Research Institute > ICND 210 > 10550 N. Torrey Pines Road > La Jolla, CA 92037 > (858) 784-8437 > [hidden email] > > * * * * * * * * * * * Prof Ian Gibbins Anatomy & Histology Flinders University GPO Box 2100 Adelaide SA 5001 AUSTRALIA [hidden email] voice: +61-8-8204 5271 fax: +61-8-8277 0085 http://som.flinders.edu.au/FUSA/Anatomy/ http://www.flinders.edu.au/neuroscience |
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In reply to this post by Kevin Braeckmans
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal At 08:39 07-08-2008, <[hidden email]> wrote: >Colocalisation based on the coincidence of colors in >still images is always limited by the spatial resolution. Keep in mind though that resolution is commonly defined as the minimum distance at which to objects of the same color in an image can be discerned. However, the relative position of objects of different colors can be determined with much higher precision because their intensity profiles can be analyzed individually (provided that the S/N is sufficient and that chromatic abberrations of the system are accounted for). Beat |
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