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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