C. elegans imaging
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** We have a lab that wants to screen a C. Elegans genetic library with about 18,000 genes. They want to take images of the worms on 24 well plates and measure the distance between 2 of 3 EGFP spots on the worms. We would like to do the experiments on an inverted HCS automated microscope. The problem is they have the worms growing on a ~1 mm thick nutrient-rich agar layer. We need to image the worms through the agar so we need to find a different way to prepare the samples. Has anyone ever tried something like this? If so do you have a solution for this? The agar is poured by hand so getting it much thinner than 1 mm would be difficult. Sincerely, Claire -------------------------------------------------------------------------------- Claire M. Brown, PhD McGill University Life Sciences Complex Imaging Facility Director |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Claire, It depends on the working distance of the objective you are using. Some objectives can focus on a specimen a few millimetres away from the lens, so 1 mm agar on top of a plastic plate wouldn't be a problem. But of course you'll have reduced NA for longer WD. It also depends how they prepare samples. Do they need to image moving worms? If so, they will need some kind of worm-tracking system which could complicate things more. If the worms are somehow paralyzed for imaging, then I don't see any reason why agar has to be used. You can keep them in liquid for a while. In my case, I place worms in between a cover slip and thin agarose layer to minimize the working distance and disturbance of worm's locomotion. With this setup, relatively high NA objective (0.75) can be used for brighter images of moving worms. One other note; I use agarose instead of agar because agar has too much auto-fluorescence. Best, Ippei (11/09/23 21:37), > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > > We have a lab that wants to screen a C. Elegans genetic library with about 18,000 genes. They want to take images of the worms on 24 well plates and measure the distance between 2 of 3 EGFP spots on the worms. > > We would like to do the experiments on an inverted HCS automated microscope. The problem is they have the worms growing on a ~1 mm thick nutrient-rich agar layer. > > We need to image the worms through the agar so we need to find a different way to prepare the samples. Has anyone ever tried something like this? If so do you have a solution for this? The agar is poured by hand so getting it much thinner than 1 mm would be difficult. > > > Sincerely, > > Claire > > -------------------------------------------------------------------------------- > Claire M. Brown, PhD > McGill University > Life Sciences Complex Imaging Facility Director > -- Ippei Kotera, PhD Centre for Research in Neurodegenerative Diseases Tanz Neuroscience Building, Room 221 University of Toronto 6 Queen's Park Crescent West Toronto, Ontario M5S 3H2 Phone: (416)978-2503 Fax: (416)978-1878 |
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In reply to this post by Claire Brown
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** 1 mm agar plus a 1mm+ plastic base are not ideal imaging conditions. A long working distance objective will focus but the thick plastic base will degrade the image - most objectives are designed for 0.17mm glass. But how good do the images need to be for your purposes ? A solution is glass bottomed multiwell plates, but they are relatively expensive. Given the popularity of multiwell plates for imaging there is probably a decent market for a plate with a suitably thin plastic bottom. Quoting "Claire Brown, Dr." <[hidden email]>: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > > We have a lab that wants to screen a C. Elegans genetic library with > about 18,000 genes. They want to take images of the worms on 24 well > plates and measure the distance between 2 of 3 EGFP spots on the > worms. > > We would like to do the experiments on an inverted HCS automated > microscope. The problem is they have the worms growing on a ~1 mm > thick nutrient-rich agar layer. > > We need to image the worms through the agar so we need to find a > different way to prepare the samples. Has anyone ever tried > something like this? If so do you have a solution for this? The agar > is poured by hand so getting it much thinner than 1 mm would be > difficult. > > > Sincerely, > > Claire > > -------------------------------------------------------------------------------- > Claire M. Brown, PhD > McGill University > Life Sciences Complex Imaging Facility Director > Jeremy Adler IGP Rudbeckslaboratoriet Daghammersköljdsväg 20 751 85 Uppsala Sweden 0046 (0)18 471 4607 |
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In reply to this post by Claire Brown
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** This paper (or the references within) may be relevant to this question. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2980495/ (or at least you can contact the author and ask what they did) Monica On Fri, Sep 23, 2011 at 9:37 PM, Claire Brown, Dr. <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > > We have a lab that wants to screen a C. Elegans genetic library with about > 18,000 genes. They want to take images of the worms on 24 well plates and > measure the distance between 2 of 3 EGFP spots on the worms. > > We would like to do the experiments on an inverted HCS automated > microscope. The problem is they have the worms growing on a ~1 mm thick > nutrient-rich agar layer. > > We need to image the worms through the agar so we need to find a different > way to prepare the samples. Has anyone ever tried something like this? If so > do you have a solution for this? The agar is poured by hand so getting it > much thinner than 1 mm would be difficult. > > > Sincerely, > > Claire > > > -------------------------------------------------------------------------------- > Claire M. Brown, PhD > McGill University > Life Sciences Complex Imaging Facility Director > |
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In reply to this post by Claire Brown
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Claire, Currently we use optical bottom 96 or 384 well plates for our HCS imaging of C. elegans but we have the luxury of the worm biosort to dispense the worms into the wells. Another way we have been doing screens is to wash the worms off the plate with PBS + o.o1% triton x-100 (so the worms don't stick to the side of pipet tips etc.) let them settle on ice then manually dispense into wells of the optical bottom plates. Current anesthesia we use is 0.05M NaAz or 1mM levamisole. If you want to keep the on the 24 well plates then you could try using more NGM filling the well up and the using optical sealing film and turn the plate over. Not great for bright field but an option, however they will move like crazy. The woes of C. elegans imaging. Let me know if I can be anymore help Cheers Cliff Cliff Luke, Ph.D. Assistant Professor, University of Pittsburgh, Department of Pediatrics, Childrens Hospital of Pittsburgh One Childrens Hospital Drive 4401 Penn Avenue Rangos, Rm 7131 Pittsburgh, PA 15224 USA Tel: +1 412 692 9456 Fax: +1 412 692 8906 email: [hidden email] On Sat, 24 Sep 2011 01:37:24 +0000, Claire Brown, Dr. <[hidden email]> wrote: >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > > >We have a lab that wants to screen a C. Elegans genetic library with about 18,000 genes. They want to take images of the worms on 24 well plates and measure the distance between 2 of 3 EGFP spots on the worms. > >We would like to do the experiments on an inverted HCS automated microscope. The problem is they have the worms growing on a ~1 mm thick nutrient-rich agar layer. > >We need to image the worms through the agar so we need to find a different way to prepare the samples. Has anyone ever tried something like this? If so do you have a solution for this? The agar is poured by hand so getting it much thinner than 1 mm would be difficult. > > >Sincerely, > >Claire > >-------------------------------------------------------------------------------- >Claire M. Brown, PhD >McGill University >Life Sciences Complex Imaging Facility Director |
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In reply to this post by Claire Brown
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Claire, Definitely use glass bottom plates, but they are expensive. We use them for screening zebrafish with 10X/.4 or .5 (3 mm wd) and 20X/.75 lenses (650 um wd). We re-used them many times after bleaching. Maybe a hot water treatment to remove the agarose. Can defined amounts agarose be added by a multi-well pipettor to better control thickness? If you are restricted to a ~1 mm agarose thickness, the maybe a high mag, low NA lens or a place a doubler in front of the camera to achieve Nyquist. 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] On Sep 23, 2011, at 6:37 PM, Claire Brown, Dr. wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > > We have a lab that wants to screen a C. Elegans genetic library with about 18,000 genes. They want to take images of the worms on 24 well plates and measure the distance between 2 of 3 EGFP spots on the worms. > > We would like to do the experiments on an inverted HCS automated microscope. The problem is they have the worms growing on a ~1 mm thick nutrient-rich agar layer. > > We need to image the worms through the agar so we need to find a different way to prepare the samples. Has anyone ever tried something like this? If so do you have a solution for this? The agar is poured by hand so getting it much thinner than 1 mm would be difficult. > > > Sincerely, > > Claire > > -------------------------------------------------------------------------------- > Claire M. Brown, PhD > McGill University > Life Sciences Complex Imaging Facility Director |
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