http://confocal-microscopy-list.275.s1.nabble.com/XYZ-drifts-giving-advice-and-looking-for-advice-tp5977803p5985631.html
image digitally post-acquisition, depending on the Z-shifts. This is
low-signal vesicles moving through the cell. So oversampling would cause
>*****
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>
>From a software side you can also just image volumes and then use image
>registration to undo the drifts in the data. This may not be optimal for
>your application though.
>
>Craig
>
>
>On Mon, Jan 31, 2011 at 10:22 AM, Craig Brideau <
[hidden email]>wrote:
>
>> 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:
>>
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>>> 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
>>>
>>
>>