*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Daniel, 

A while back I solved a similar problem by eliminating vibrations on the table.  We moved the worst offenders (e.g., fans) off the table and putting vibration-absorbing rubber under everything that could not be moved.  Bits of carpet padding work well for that.  It would also be a good idea to find out how stable the climate control is in your room.  A mercury bulb and gas laser can really warm up a room with slow air handling.  Finally, have you checked whether your water miniscus is evaporating?  It could go pretty fast at 37 c. This is basic stuff that you most likely checked already; if not it might help.  

cheers, 


Tim Feinstein


On Jan 31, 2011, at 11:49 AM, Daniel Murphy wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> 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