Z-Drift-LSM510
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello All, I am seeing this problem recently on our LSM510. During a timelapse, the specimen is going out of focus (drifting down). Initially I thought it maybe because of the 40X water lens but its happening with the 40X oil and 20x lens too (so I assume it maybe true with 10X also-not tried that yet). The scope is equipped with a XL-3 incubator and temp is constant at 37deg. I have let the Argon laser warm up for 45mins to 1hr before the timelapse and it still happens. I have tried imaging with 2P chameleon and it still happens. Anyone experienced this problem before? Is this temperature related or the focus board on the scope is about to die? Any suggestions appreciated. Thank you very much. Abhi Abhijit Bugde, M.S Research Associate, Cell Biology Live Cell Imaging Core Facility UT Southwestern Medical Center 5323 Harry Hines Blvd. Dallas, TX 75390-9039 T: 214-648-9935 |
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Search the CONFOCAL archive at
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Abhijit,
Almost always, drift is caused by a temperature dis-equilibrium. Can you warm up your XL-3 chamber overnight before imaging? That usually fixes the drift problem on our XL-3, which does not have a focus board. Also, we never put the heating unit knob past the middle position. Takes longer to warm up, but does so more evenly. Of course, an overnight warmup may not be compatible with a with multi-user core system. MIke On May 16, 2008, at 5:38 PM, Abhijit Bugde wrote:
Michael J. Schell, Ph.D., CIV, USUHS Assist. Professor Dept. of Pharmacology Uniformed Services University 4301 Jones Bridge Rd. Bethesda, MD 20814-3220 tel: (301) 295-3249 |
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In reply to this post by Abhijit Bugde
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Abhi, I had the same problem before. I realized that the wire connected to the stage was too tight in the hole of the incubator, causing strain on the stage and z-drift. Thus, just before starting the timelapse, I make sure that the wire do not produce any pressure on the stage, allowing 24-hours timelapses without any z-drift. Good luck, Nicolas -----Original Message----- From: Abhijit Bugde [mailto:[hidden email]] Sent: May 16, 2008 5:38 PM To: [hidden email] Subject: Z-Drift-LSM510 Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello All, I am seeing this problem recently on our LSM510. During a timelapse, the specimen is going out of focus (drifting down). Initially I thought it maybe because of the 40X water lens but its happening with the 40X oil and 20x lens too (so I assume it maybe true with 10X also-not tried that yet). The scope is equipped with a XL-3 incubator and temp is constant at 37deg. I have let the Argon laser warm up for 45mins to 1hr before the timelapse and it still happens. I have tried imaging with 2P chameleon and it still happens. Anyone experienced this problem before? Is this temperature related or the focus board on the scope is about to die? Any suggestions appreciated. Thank you very much. Abhi Abhijit Bugde, M.S Research Associate, Cell Biology Live Cell Imaging Core Facility UT Southwestern Medical Center 5323 Harry Hines Blvd. Dallas, TX 75390-9039 T: 214-648-9935 |
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In reply to this post by Michael Schell
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The objective itself is also a tremendous heat sink, although if you haven't seen this drifting problem in the past that may not be your problem.
Craig On Fri, May 16, 2008 at 3:58 PM, Michael Schell <[hidden email]> wrote:
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In reply to this post by Abhijit Bugde
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello All, Thank you very much for all the replies. I will try to answer most of the questions in 1 post. -I havent checked if the wires are causing any strain on the stage. Hope its as simple as that for me too. -We are using the EXFO lamp so its not heating the stand. -I have kept the incubator at a steady temp overnight but that does not solve the drift problem. -Always used the turret loaded with objectives but will try to image with 1 objective to find out if its gravity causing the problem. Again, thank you for the feedback. Best regards, Abhi |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have been struggling with the same problem for a long time. End up correcting the shift manually between imaging points, since there was always a drift in same direction (approximately 2-3µm/h). Not the most elegant way but it worked out well. Could not find another solution until now (unfortunately including suggestions that were given here already). There might be a software solution for the Zeiss imaging system at http://www.zeiss.de/lsm-macros (see multiple time series) I have never tried it though. Good luck, Andreas >---------------------------------------------- > Andreas Vlachos > Neuroscience Center > Institute of Clinical Neuroanatomy > Goethe-University Frankfurt > Heinrich-Hofmann street 7 > D-60590 Frankfurt am Main, Germany > Phone: ++49 (69) 6301 83323 > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Abhijit Bugde > Sent: Samstag, 17. Mai 2008 05:27 > To: [hidden email] > Subject: Re: Z-Drift-LSM510 > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello All, > > Thank you very much for all the replies. I will try to answer most of > the > questions in 1 post. > > -I havent checked if the wires are causing any strain on the stage. > Hope > its as simple as that for me too. > > -We are using the EXFO lamp so its not heating the stand. > > -I have kept the incubator at a steady temp overnight but that does not > solve the drift problem. > > -Always used the turret loaded with objectives but will try to image > with 1 > objective to find out if its gravity causing the problem. > > Again, thank you for the feedback. > > Best regards, > Abhi |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Have you tried to remove all other objectives? We found that cures the problem on our Applied Precision DeltaVision Systems which are based on the IX-71 OLYPUS. It also has a tension ring which we set to strong. Good luck Hugo Ostermann CHROMAPHOR Analysen-Technik GmbH Mühlenkamp 37 D-59387 Ascheberg - Germany Tel: ++49 - 2593-928.600 Fax: ++49 - 2593-928.601 mail: [hidden email] URL: http://www.chromaphor.de Sitz Ascheberg-Westfalen Amtsgericht - Registergericht Coesfeld HRB 7703 Geschäftsführer: Hugo Ostermann -----Ursprüngliche Nachricht----- Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Andreas V Gesendet: Samstag, 17. Mai 2008 11:53 An: [hidden email] Betreff: Re: Z-Drift-LSM510 Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have been struggling with the same problem for a long time. End up correcting the shift manually between imaging points, since there was always a drift in same direction (approximately 2-3µm/h). Not the most elegant way but it worked out well. Could not find another solution until now (unfortunately including suggestions that were given here already). There might be a software solution for the Zeiss imaging system at http://www.zeiss.de/lsm-macros (see multiple time series) I have never tried it though. Good luck, Andreas >---------------------------------------------- > Andreas Vlachos > Neuroscience Center > Institute of Clinical Neuroanatomy > Goethe-University Frankfurt > Heinrich-Hofmann street 7 > D-60590 Frankfurt am Main, Germany > Phone: ++49 (69) 6301 83323 > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Abhijit Bugde > Sent: Samstag, 17. Mai 2008 05:27 > To: [hidden email] > Subject: Re: Z-Drift-LSM510 > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello All, > > Thank you very much for all the replies. I will try to answer most of > the > questions in 1 post. > > -I havent checked if the wires are causing any strain on the stage. > Hope > its as simple as that for me too. > > -We are using the EXFO lamp so its not heating the stand. > > -I have kept the incubator at a steady temp overnight but that does not > solve the drift problem. > > -Always used the turret loaded with objectives but will try to image > with 1 > objective to find out if its gravity causing the problem. > > Again, thank you for the feedback. > > Best regards, > Abhi |
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In reply to this post by Abhijit Bugde
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Abhi - The XL3 incubator is only enclosing part of the microscope and metal has a high heat conductance, so changes in room temperature may still cause focus drift. (That's why we design our incubators to enclose as much of the microscope as possible => attention, commercial interest). Monitor the ambient temperature to see if there is a correlation with focus instability. That said, I want to add that there are also other reasons for focus instability: I presume that you have already checked that the temperature inside the incubator is really stable. Fluctuations as small as 0.3°C can cause clearly noticeable focus variations. Then keep in mind that not just the microscope but also the sample needs to be thermally equilibrated. That can take quite some time, too (up to several hours, depending on the sample format). With today's research-grade microscopes, mechanical slip is much less common than with their predecessors, but it is still worth checking for. A good indicator is a monotonous drift following gravity. With open "elastic" sample carriers (e.g. plastic dishes), weight reduction due to evaporation of culture medium can have the opposite effect, i.e. monotonous drift against gravity. If that is the case, you should see no drift with fixed embedded samples. With piezo or galvo Z-drives, electrical drift is also a candidate. Check if you still see drift if you de-power or remove the device. Last but not least, variations in heat dissipation of electrical devices (e.g. a motorized stage) can cause local thermal fluctuations that cannot be compensated by the incubator. Again, de-power the device in question to check if it is the culprit. Best regards, Beat Life Imaging Services At 05:26 17-05-2008, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hello All, > >Thank you very much for all the replies. I will try to answer most of the >questions in 1 post. > >-I havent checked if the wires are causing any strain on the stage. Hope >its as simple as that for me too. > >-We are using the EXFO lamp so its not heating the stand. > >-I have kept the incubator at a steady temp overnight but that does not >solve the drift problem. > >-Always used the turret loaded with objectives but will try to image with 1 >objective to find out if its gravity causing the problem. > >Again, thank you for the feedback. > >Best regards, >Abhi |
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Search the CONFOCAL archive at
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Abhi,
The advices given so far assume that the drift is not caused by a real displacement of the Z motor of your scope. However, I recently had the problem of a faulty encoder inside the Z knob of the touchscreen remote control unit of a Zeiss Axio Observer, causing the focus to change in a non-reproducible maner (sometimes as bad as 1 micron/minute). The ironic part is that I spent some time checking temperature, vibration, etc... before realizing that the drift was actually monitored by the Z motor position, indicating a motor problem. So far the Zeiss people have changed the touchscreen unit two times, and the interface card inside the scope, and the problem is attenuated but still present. The solutions are to record the Z position and force to re-actuate it before each images, or just unplug the touchscreen unit before launching a timelapse (altough it may be not so good to "hot" unplug the unit). So I'd suggest you check first if the drift is recorded by your stage/Z motor position (on the scope or inside the software you use), and if re-actuating the Z-position to a recorded point can compensate for the drift. If not, then the problems are thermal instability or mechanical problems such as vibrations as people pointed out previouly. Christophe On Sat, May 17, 2008 at 9:23 PM, Beat Ludin <[hidden email]> wrote: Abhi - |
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