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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