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All:

Thanks to all for the helpful and thorough advice.  From your comments, we
definitely have some ideas to work through to nail down the problem-causing
element(s).  From your comments and after speaking with a tech from Zeiss,
apparently this is an issue that previous users have also had with long
time-lapse imaging of incubated samples on the Axiovert 200M microscope.  He
had done previous studies and found that with a similar setup, except with
40x dry objective (instead of 40x water), there is a Z drift of about 13um
in the first 6 hours of turning the system on, at which point the Z drift is
more or less gone.  So for really stable time-lapse work, the system needs
to be on for at least 6hrs!  This is without an incubator enclosing the
entire scope like the Zeiss XL --- perhaps this would reduce the
equilibration time (any thoughts/experience?).

What solutions are there to the problem of the water evaporating off the
objective?  We use a water substitute with the same n but it is a little
thicker, and so less evaporation.

Thanks!

Dan

On Tue, 1 Feb 2011 13:51:12 -0000, Keith Morris <[log in to unmask]> wrote:

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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:� [log in to unmask]
>Web-pages: http://www.well.ox.ac.uk/molecular-cytogenetics-and-microscopy
>
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[log in to unmask]] On
>Behalf Of Danielle Crippen
>Sent: 31 January 2011 17:51
>To: [log in to unmask]
>Subject: Re: XYZ drifts - giving advice and looking for advice
>
>*****
>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:[log in to unmask]] On
>Behalf Of Ramshesh, Venkat K
>Sent: Monday, January 31, 2011 9:41 AM
>To: [log in to unmask]
>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: [log in to unmask]
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[log in to unmask]] On
>Behalf Of Daniel Murphy
>Sent: Monday, January 31, 2011 11:50 AM
>To: [log in to unmask]
>Subject: XYZ drifts - giving advice and looking for advice
>
>*****
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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