stage drift, or not?
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Cromey, Douglas W - (dcromey) |
stage drift, or not?
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Confocal colleagues, Last week I had a user complain of excessive Z-drift in the stage of our upright LSM510. Her sample is a small conical piece of renal tissue mounted in agar in a 50mm plastic petri dish. The goal is to optically section as deeply as possible and observe the differently stained renal tubules. I watched her set things up and I can confirm that the drift with her sample is on the order of 10s of microns in 1-2 minutes. Zeiss service requested I test a bead slide for Z-drift before sending in a service engineer. I took an image every 30s for 21 minutes and saw no appreciable drift. Both Zeiss and I have suggested to the user that the problem is sample-related. The user is not completely happy with this suggestion. Any other possibilities that you can think of? Doug ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of Cellular & Molecular Medicine, University of Arizona 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA office: AHSC 4212 email: [hidden email] voice: 520-626-2824 fax: 520-626-2097 http://swehsc.pharmacy.arizona.edu/exppath/ Home of: "Microscopy and Imaging Resources on the WWW" |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I see drift that lasts about 30 minutes and then slows down and stops. In my case, I suspect the piezo is rebounding after the pressure of placing the specimen on the stage. On Mon, Sep 10, 2012 at 9:40 AM, Cromey, Douglas W - (dcromey) <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Confocal colleagues, > > Last week I had a user complain of excessive Z-drift in the stage of our upright LSM510. Her sample is a small conical piece of renal tissue mounted in agar in a 50mm plastic petri dish. The goal is to optically section as deeply as possible and observe the differently stained renal tubules. I watched her set things up and I can confirm that the drift with her sample is on the order of 10s of microns in 1-2 minutes. > > Zeiss service requested I test a bead slide for Z-drift before sending in a service engineer. I took an image every 30s for 21 minutes and saw no appreciable drift. > > Both Zeiss and I have suggested to the user that the problem is sample-related. The user is not completely happy with this suggestion. > > Any other possibilities that you can think of? > > Doug > > ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ > Douglas W. Cromey, M.S. - Associate Scientific Investigator > Dept. of Cellular & Molecular Medicine, University of Arizona > 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA > > office: AHSC 4212 email: [hidden email] > voice: 520-626-2824 fax: 520-626-2097 > > http://swehsc.pharmacy.arizona.edu/exppath/ > Home of: "Microscopy and Imaging Resources on the WWW" -- Michael J. Herron, U of MN, Dept. of Entomology [hidden email] 612-624-3688 (office) 612-625-5299 (FAX) |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Is the renal tissue and agar originally incubated at 37C? This is probably thermal shift. Putting beads on the stage s not a good comparison, unless you incubate the beads at 37C and then put them on. -- Teng-Leong Chew, PhD Director, Cell Imaging Facility & Nikon Imaging Center Northwestern University 312-503-2841 On 9/10/12 9:46 AM, "Michael Herron" <[hidden email]> wrote: >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >I see drift that lasts about 30 minutes and then slows down and stops. >In my case, I suspect the piezo is rebounding after the pressure of >placing the specimen on the stage. > > > >On Mon, Sep 10, 2012 at 9:40 AM, Cromey, Douglas W - (dcromey) ><[hidden email]> wrote: >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Confocal colleagues, >> >> Last week I had a user complain of excessive Z-drift in the stage of >>our upright LSM510. Her sample is a small conical piece of renal tissue >>mounted in agar in a 50mm plastic petri dish. The goal is to optically >>section as deeply as possible and observe the differently stained renal >>tubules. I watched her set things up and I can confirm that the drift >>with her sample is on the order of 10s of microns in 1-2 minutes. >> >> Zeiss service requested I test a bead slide for Z-drift before sending >>in a service engineer. I took an image every 30s for 21 minutes and saw >>no appreciable drift. >> >> Both Zeiss and I have suggested to the user that the problem is >>sample-related. The user is not completely happy with this suggestion. >> >> Any other possibilities that you can think of? >> >> Doug >> >> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ >> Douglas W. Cromey, M.S. - Associate Scientific Investigator >> Dept. of Cellular & Molecular Medicine, University of Arizona >> 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA >> >> office: AHSC 4212 email: [hidden email] >> voice: 520-626-2824 fax: 520-626-2097 >> >> http://swehsc.pharmacy.arizona.edu/exppath/ >> Home of: "Microscopy and Imaging Resources on the WWW" > > > >-- >Michael J. Herron, U of MN, Dept. of Entomology > [hidden email] > 612-624-3688 (office) 612-625-5299 (FAX) |
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Vladimir Ghukasyan-2 |
Re: stage drift, or not?
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In reply to this post by Michael Herron
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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 Douglas, I assume that your testing slide had beads dried and then covered with a mounting media. In this condition they're much more prone to vibrations than a sample in agar. Try to make a sample with fluorescent beads in agar, then your testing will be comparable. With regards, Vladimir On Mon, Sep 10, 2012 at 10:46 AM, Michael Herron <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > I see drift that lasts about 30 minutes and then slows down and stops. > In my case, I suspect the piezo is rebounding after the pressure of > placing the specimen on the stage. > > > > On Mon, Sep 10, 2012 at 9:40 AM, Cromey, Douglas W - (dcromey) > <[hidden email]> wrote: >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Confocal colleagues, >> >> Last week I had a user complain of excessive Z-drift in the stage of our upright LSM510. Her sample is a small conical piece of renal tissue mounted in agar in a 50mm plastic petri dish. The goal is to optically section as deeply as possible and observe the differently stained renal tubules. I watched her set things up and I can confirm that the drift with her sample is on the order of 10s of microns in 1-2 minutes. >> >> Zeiss service requested I test a bead slide for Z-drift before sending in a service engineer. I took an image every 30s for 21 minutes and saw no appreciable drift. >> >> Both Zeiss and I have suggested to the user that the problem is sample-related. The user is not completely happy with this suggestion. >> >> Any other possibilities that you can think of? >> >> Doug >> >> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ >> Douglas W. Cromey, M.S. - Associate Scientific Investigator >> Dept. of Cellular & Molecular Medicine, University of Arizona >> 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA >> >> office: AHSC 4212 email: [hidden email] >> voice: 520-626-2824 fax: 520-626-2097 >> >> http://swehsc.pharmacy.arizona.edu/exppath/ >> Home of: "Microscopy and Imaging Resources on the WWW" > > > > -- > Michael J. Herron, U of MN, Dept. of Entomology > [hidden email] > 612-624-3688 (office) 612-625-5299 (FAX) |
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In reply to this post by Michael Herron
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I've seen something similar when imaging really thick brain tissues - they tend to get squished at the top of z-scan when using an immersion objective, probably due to pressure waves caused by the scanning motion of the z-piezo. Maybe switch to a mounting medium that hardens instead of something soft like agar? You could also try sprinkling your agar with fluorescent beads as well to see if the z-drift is worse in the agar-based sample. John Oreopoulos Research Assistant Spectral Applied Research Richmond Hill, Ontario Canada www.spectral.ca On 2012-09-10, at 10:46 AM, Michael Herron wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > I see drift that lasts about 30 minutes and then slows down and stops. > In my case, I suspect the piezo is rebounding after the pressure of > placing the specimen on the stage. > > > > On Mon, Sep 10, 2012 at 9:40 AM, Cromey, Douglas W - (dcromey) > <[hidden email]> wrote: >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Confocal colleagues, >> >> Last week I had a user complain of excessive Z-drift in the stage of our upright LSM510. Her sample is a small conical piece of renal tissue mounted in agar in a 50mm plastic petri dish. The goal is to optically section as deeply as possible and observe the differently stained renal tubules. I watched her set things up and I can confirm that the drift with her sample is on the order of 10s of microns in 1-2 minutes. >> >> Zeiss service requested I test a bead slide for Z-drift before sending in a service engineer. I took an image every 30s for 21 minutes and saw no appreciable drift. >> >> Both Zeiss and I have suggested to the user that the problem is sample-related. The user is not completely happy with this suggestion. >> >> Any other possibilities that you can think of? >> >> Doug >> >> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ >> Douglas W. Cromey, M.S. - Associate Scientific Investigator >> Dept. of Cellular & Molecular Medicine, University of Arizona >> 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA >> >> office: AHSC 4212 email: [hidden email] >> voice: 520-626-2824 fax: 520-626-2097 >> >> http://swehsc.pharmacy.arizona.edu/exppath/ >> Home of: "Microscopy and Imaging Resources on the WWW" > > > > -- > Michael J. Herron, U of MN, Dept. of Entomology > [hidden email] > 612-624-3688 (office) 612-625-5299 (FAX) |
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In reply to this post by Cromey, Douglas W - (dcromey)
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear Doug Is the tissue/agarose covered in some liquid of some sort? If not, it will shrink as it dries. I did loads of agarose shrinking experiments in the past... :( Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader Live Cell Imaging Unit Dept of Biosciences and Nutrition Karolinska Institutet Novum 14183 Huddinge Sweden office: +46 (0) 8 5248 1107 LCI room: +46 (0) 8 5248 1172 mobile: +46 (0) 73 733 5008 > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]] On Behalf Of Cromey, > Douglas W - (dcromey) > Sent: 10 September 2012 16:41 > To: [hidden email] > Subject: stage drift, or not? > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Confocal colleagues, > > Last week I had a user complain of excessive Z-drift in the stage of our upright > LSM510. Her sample is a small conical piece of renal tissue mounted in agar in a > 50mm plastic petri dish. The goal is to optically section as deeply as possible and > observe the differently stained renal tubules. I watched her set things up and I can > confirm that the drift with her sample is on the order of 10s of microns in 1-2 > minutes. > > Zeiss service requested I test a bead slide for Z-drift before sending in a service > engineer. I took an image every 30s for 21 minutes and saw no appreciable drift. > > Both Zeiss and I have suggested to the user that the problem is sample-related. > The user is not completely happy with this suggestion. > > Any other possibilities that you can think of? > > Doug > > ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ > Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of Cellular & > Molecular Medicine, University of Arizona > 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA > > office: AHSC 4212 email: [hidden email] > voice: 520-626-2824 fax: 520-626-2097 > > http://swehsc.pharmacy.arizona.edu/exppath/ > Home of: "Microscopy and Imaging Resources on the WWW" |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Agar, collagen matrix, etc may expand or shrink with changes in hydration. We have stopped evaporation in samples by smothering them in tissue culture grade mineral oil used for culturing mouse embryos. CO2 goes through fine, water vapor doesn't, and I don't know about other gasses. -Michael C. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sylvie Le Guyader Sent: Monday, September 10, 2012 11:00 AM To: [hidden email] Subject: Re: stage drift, or not? ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear Doug Is the tissue/agarose covered in some liquid of some sort? If not, it will shrink as it dries. I did loads of agarose shrinking experiments in the past... :( Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader Live Cell Imaging Unit Dept of Biosciences and Nutrition Karolinska Institutet Novum 14183 Huddinge Sweden office: +46 (0) 8 5248 1107 LCI room: +46 (0) 8 5248 1172 mobile: +46 (0) 73 733 5008 > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]] On Behalf Of Cromey, Douglas > W - (dcromey) > Sent: 10 September 2012 16:41 > To: [hidden email] > Subject: stage drift, or not? > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Confocal colleagues, > > Last week I had a user complain of excessive Z-drift in the stage of > our upright LSM510. Her sample is a small conical piece of renal > tissue mounted in agar in a 50mm plastic petri dish. The goal is to > optically section as deeply as possible and observe the differently > stained renal tubules. I watched her set things up and I can confirm > that the drift with her sample is on the order of 10s of microns in 1-2 minutes. > > Zeiss service requested I test a bead slide for Z-drift before sending > in a service engineer. I took an image every 30s for 21 minutes and saw no appreciable drift. > > Both Zeiss and I have suggested to the user that the problem is sample-related. > The user is not completely happy with this suggestion. > > Any other possibilities that you can think of? > > Doug > > ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ > Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of > Cellular & Molecular Medicine, University of Arizona > 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA > > office: AHSC 4212 email: [hidden email] > voice: 520-626-2824 fax: 520-626-2097 > > http://swehsc.pharmacy.arizona.edu/exppath/ > Home of: "Microscopy and Imaging Resources on the WWW" |
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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've had the situation where the sample sticks a bit to our immersion lens, so as we do our Z-stack the sample gets pulled up or pushed down by the lens. It seems just the adhesion of the water between sample and objective can be enough for this. We net our samples down to prevent this. Craig On Mon, Sep 10, 2012 at 9:46 AM, Cammer, Michael <[hidden email] > wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Agar, collagen matrix, etc may expand or shrink with changes in hydration. > We have stopped evaporation in samples by smothering them in tissue > culture grade mineral oil used for culturing mouse embryos. CO2 goes > through fine, water vapor doesn't, and I don't know about other gasses. > -Michael C. > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Sylvie Le Guyader > Sent: Monday, September 10, 2012 11:00 AM > To: [hidden email] > Subject: Re: stage drift, or not? > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Dear Doug > > Is the tissue/agarose covered in some liquid of some sort? If not, it will > shrink as it dries. I did loads of agarose shrinking experiments in the > past... :( > > Med vänlig hälsning / Best regards > > Sylvie > > @@@@@@@@@@@@@@@@@@@@@@@@ > Sylvie Le Guyader > Live Cell Imaging Unit > Dept of Biosciences and Nutrition > Karolinska Institutet > Novum > 14183 Huddinge > Sweden > office: +46 (0) 8 5248 1107 > LCI room: +46 (0) 8 5248 1172 > mobile: +46 (0) 73 733 5008 > > > -----Original Message----- > > From: Confocal Microscopy List > > [mailto:[hidden email]] On Behalf Of Cromey, Douglas > > W - (dcromey) > > Sent: 10 September 2012 16:41 > > To: [hidden email] > > Subject: stage drift, or not? > > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > ***** > > > > Confocal colleagues, > > > > Last week I had a user complain of excessive Z-drift in the stage of > > our upright LSM510. Her sample is a small conical piece of renal > > tissue mounted in agar in a 50mm plastic petri dish. The goal is to > > optically section as deeply as possible and observe the differently > > stained renal tubules. I watched her set things up and I can confirm > > that the drift with her sample is on the order of 10s of microns in 1-2 > minutes. > > > > Zeiss service requested I test a bead slide for Z-drift before sending > > in a service engineer. I took an image every 30s for 21 minutes and saw > no appreciable drift. > > > > Both Zeiss and I have suggested to the user that the problem is > sample-related. > > The user is not completely happy with this suggestion. > > > > Any other possibilities that you can think of? > > > > Doug > > > > ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ > > Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of > > Cellular & Molecular Medicine, University of Arizona > > 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA > > > > office: AHSC 4212 email: [hidden email] > > voice: 520-626-2824 fax: 520-626-2097 > > > > http://swehsc.pharmacy.arizona.edu/exppath/ > > Home of: "Microscopy and Imaging Resources on the WWW" > |
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