http://confocal-microscopy-list.275.s1.nabble.com/XYZ-drifts-giving-advice-and-looking-for-advice-tp5977803p5977903.html
registration to undo the drifts in the data. This may not be optimal for
> Box in your scope; that is, build an enclosure around it. The box will
> block any air currents or other disturbances. If temperature drift in the
> room is more than 1 degree C you can also add temperature control to the box
> to thermally isolate the microscope from the room. Finally, what sort of
> table is the microscope sitting on?
>
> Craig
>
>
> On Mon, Jan 31, 2011 at 9:49 AM, Daniel Murphy <
>
[hidden email]> 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
>>
>
>