http://confocal-microscopy-list.275.s1.nabble.com/XYZ-drifts-giving-advice-and-looking-for-advice-tp5977803p5985825.html
There is most likely some variability between systems. Our Axiovert 200M was installed with an ASI 2000 motorized stage. Overnight timelapses with a brightfield specimen (fixed, stable) displayed several microns of drift in x,y and z using a 40X/.75 air objective. Building an enclosure reduced this drift dramatically, and heating to 37C eliminated it entirely after 3 hours of pre-heating.
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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 <
[hidden email]> 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:�
[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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>>
>> 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
>>
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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