http://confocal-microscopy-list.275.s1.nabble.com/XYZ-drifts-giving-advice-and-looking-for-advice-tp5977803p5985769.html
Hi Dan, we have the same instrument and objective and have the same problem (Zeiss LSM510 inverted and 40x/1.2W). To get around this problem we purchased a new scope with the entire scope enclosed in the incubator, just as described by Kieth Morris, and I agree completely with his advice. The large XL incubator almost completely removes the focus drift problem, if the incubator is turned on around 2 hours prior to image acquisition. I was surprised and impressed by the difference the large incubator makes. As already described; the cost of the incubator system is really in the components and not the box so we purchased the Pecon acrylic box which is really quite nice to have as it is see-through.
Back to your immediate problem, as you already point out, if you turn on the small Pecon incubator at least two hours prior to imaging some of the focus drift problem is alleviated. We have tried the objective heater and the foam objective warmer, they are little help in my opinion. Something that did seem to help was the use of Zeiss Immersol W immersion oil for Water objectives.
Also, if you have the Zeiss multi-time macro (or MDA in AxioVision) you can pause your acquisition and refocus without stopping the experiment.
Thanks to all for the helpful and thorough advice. From your comments, we
element(s). From your comments and after speaking with a tech from Zeiss,
time-lapse imaging of incubated samples on the Axiovert 200M microscope. He
more or less gone. So for really stable time-lapse work, the system needs
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>Hi Daniel,
>
>Danielle is quite right - to reduce Z focus drift significantly a Zeiss Live
>cell large XL3 incubator is probably required on your Axiovert 200 to close
>in the entire stage, nosepiece and objectives, and even then the stage has
>be on for at least 2 hours before the time-lapse and wait 10 minutes before
>starting the timelapse after opening the incubator doors [also a bit of
>blu-tack can help to hold the Petri-dish in one place, along with any
>'mounting screw' adjustments on the heatable Labtek insert P - although you
>would probably just put your Warner unit inside the XL3 incubator]. When you
>switch on the XL3 incubator and the temperature of the objectives/stage
>rises from 22oC to 37oC you get a focus drift of 30um or more [microscopes
>are still built largely with metal rather than composites and so suffer from
>thermal expansion effects]. We used to use the bijou Zeiss on-stage PeCon
>incubator-S units similar to yours, and the Z drift was very bad for
>time-lapse [fine for viewing live cells and capturing images as you could
>refocus all the time, but not so good for time lapse, where we had to take a
>z-stack at each time-point to track the focus point]. Once the large
>incubators were fitted things improved considerably [particularly if the
>microscope is on an anti-vibration air-table].
>
>Unfortunately a Zeiss XL3 incubator system sets you back �15k+ with the
>associated PeCon electronics, precise %CO2 control and heaters. A cheaper
>option would be a Zeiss objective heater that fits on the objective under
>the stage to minimize thermal expansion effects there [but that�s still
>thousands of pounds including TempController, and probably nowhere near as
>effective as the XL3 incubator, I've never used one]. Even cheaper still
>[and even less effective but better than nothing] is to use the foam
>objective insulators Zeiss manufacture specifically for the purpose to put
>around each objective. Worse still, with an oil objective and Mattek type
>culture dish the heat is conducted away from the culture vessel into the
>cold objective. Building you own XL3 type incubator enclosure might be a bit
>cheaper if you have a workshop, but you also need the air heater control
>sorted out and our in-house incubators were still �5k each for the Perspex
>enclosure alone [as our workshop charged an hourly rate]. You also might
>need some sort of humidity control for longer timelapses [we get our water
>vapour in via the 5% CO2 feed] or some use the PeCon FoilCovers to reduce
>media evaporation with culture dishes.
>
>See the XL3 incubator
>
http://www.well.ox.ac.uk/live-cell-imaging>Zeiss/PeCon incubator options
>
http://www.pecon.biz/?page_id=55>Foil cover
>
http://www.pecon.biz/?page_id=245>
>Antivibration feedback also helps if its just the rubber door-stop feet on
>the microscope base and no air-table - but these [Z motorized] focus issues
>are mainly thermal [air conditioning and lack of a regulated heated
>enclosure surround the entire objective/stage area] - the CRUK in London go
>as far as enclosing the entire microscope within an incubator enclosure. For
>cheap antivibration control reinforce the bench and add a large heavy slab
>of granite [or sealed in concrete if poor] under the microscope and rest the
>slab on anti-vibrational pads. It worked for us [we had a workshop to do the
>mods]. Vibration can cause XY movement of the manual stage and sample
>wobbling during capture. Once these slabs were in place you could tap the
>worktop without the specimen dancing on the screen.
>
>Also try and get the room air-conditioning adjusted - the standard office
>rated lab air-con is simply balanced to 22oC [knowledge of the Victorian
>thermostat now being lost]. You can upgrade the aircon wall temperature
>sensor electronics to make it more stable/accurate [cost us �1,000],
>assuming you have one, and possibly raise the room balance temperature to
>24oC. Simple cardboard deflectors, to make sure the cool air from the aircon
>ducting doesnt blow on the microscope, can also be highly effective.
>
>Good luck with the issue, Regards
>
>Keith
>
>No commercial interest
>
>---------------------------------------------------------------------------
>Dr Keith J. Morris,
>Molecular Cytogenetics and Microscopy Core,
>Laboratory 00/069 and 00/070,
>The Wellcome Trust Centre for Human Genetics,
>Roosevelt Drive,
>Oxford� OX3 7BN,
>United Kingdom.
>
>Telephone:� +44 (0)1865 287568
>Email:�
[hidden email]
>Web-pages:
http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy>
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:
[hidden email]] On
>Behalf Of Danielle Crippen
>Sent: 31 January 2011 17:51
>To:
[hidden email]
>Subject: Re: XYZ drifts - giving advice and looking for advice
>
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>To join, leave or search the confocal microscopy listserv, go to:
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>
>We've had similar problems and have found that we need to:
>
>A. Box in the system. We've made several enclosures here...homemade ones
>are much less expensive than commercial sources (we have both) and work just
>as well...sometimes better. Write to me off list and we can talk more about
>them if you like.
>
>B. Isolate any fans in the box, so their vibration doesn't interfere with
>stage movement.
>
>C. Control the temperature and air current in the room. Even with a boxed-in
>microscope, temperature fluctuations in the surrounding environment have
>definitely caused Z drift in our experience. It has also paid off for us to
>control where the air is blowing in the room (ie. not directly down on the
>microscope)...we just used some cardboard to re-direct the air current.
>
>Best of luck!! This is a frustrating issue for sure!
>
>_______________________________
>Danielle Crippen
>Morphology and Imaging Core Manager
>Buck Institute for Research on Aging
>8001 Redwood Blvd
>Novato, CA 94945
>415-209-2046
>TheBuck.org
>
>
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:
[hidden email]] On
>Behalf Of Ramshesh, Venkat K
>Sent: Monday, January 31, 2011 9:41 AM
>To:
[hidden email]
>Subject: Re: XYZ drifts - giving advice and looking for advice
>
>Hi Dan,
>
> We had some drifts (mostly xy) on our Olympus Fluoview 1000 which were
>caused by the stage controller joystick. We had to replace the controller.
>I am not sure if this applies to you but just a thought.
>Further as Tim has already pointed out the evaporation of water meniscus
>also causes drift problems.
>
>Best,
>Venkat
>
>Venkat Ramshesh, PhD
>Bioengineer/Facility Manager
>Cell and Molecular Imaging Core
>Hollings Cancer Center and Center for Cell Death, Injury and Regeneration
>Medical University of South Carolina
>QE302
>280 Calhoun Street, MSC 140
>Charleston, SC 29425
>
>Ph: 843-792-3530
>Fax: 843-792-8436
>E-mail:
[hidden email]
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:
[hidden email]] On
>Behalf Of Daniel Murphy
>Sent: Monday, January 31, 2011 11:50 AM
>To:
[hidden email]
>Subject: XYZ drifts - giving advice and looking for advice
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>
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>
>Hello,
>
>Some advice and a plea for help on XYZ drifts!
>
>We have been doing time-lapse studies on our Zeiss LSM 5 Live, Axiovert 200M
>inverted scope. Typically these go for 20min durations at 1frame/30sec. We
>capture z-stacks that are typically 10-15 slices thick on a 40X Water with
>2.5X optical zoom. We also use a Warner instruments micro-incubation system
>DH40i (
http://www.warneronline.com/product_info.cfm?id=1165) with heated
>dish cover to maintain a temp of 37C.
>
>We have had persistent issues with drifts in X, Y and Z. Initially the
>problems were sever, with shifts of up to a few microns in all 3 directions.
> We have made progressive adjustments to our protocol with some big
>improvements, but the problem is still significant.
>
>First, we found that by surrounding the open area below the stage (where you
>can access the objectives) with seran wrap, this provided a good way of
>protecting the system from air currents and thermal influence from the
>environment. We also characterized the incubation system and found it works
>better to run it without feedback at a constant voltage (the feedback
>response was too slow because the incubation system is too much of a heat
>sink). We typically turn on the incubator and let it equilibrate for at
>least 15min (with the dish inside as well, whenever possible).
>
>XYZ drift still remains, however. It seems to come and go. For one
>experiment it will be almost unnoticeable, but for another it will make the
>data practically unusable. Sometimes the drifts are just in one direction.
> Other times the stack shifts in Z up and down several times in one time
>sequence. It seems to be very irregular and so probably due to random
>fluctuations in the environment.
>
>XY shifts are not too bad, so long as the area of interest remains in view.
> There are several ways to adjust for the shift post-acquisition --- you
>have more wiggle room since there are all those pixels in every direction.
>Z shifts are the real issue that plagues us. Any advice or ideas would be
>warmly welcomed.
>
>Daniel Murphy
>Albert Einstein College of Medicine
>Optical Imaging Manager, Cell and Molecular Neuroimaging Core 1410 Pelham
>Parkway South Bronx, NY 10461 Ph 718-430-4027/8985
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