Posted by
Keith Morris on
URL: http://confocal-microscopy-list.275.s1.nabble.com/XYZ-drifts-giving-advice-and-looking-for-advice-tp5977803p5981018.html
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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 thats 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-imagingZeiss/PeCon incubator options
http://www.pecon.biz/?page_id=55Foil cover
http://www.pecon.biz/?page_id=245Antivibration 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