Re: time series, large images and focus

Posted by Knecht, David on
URL: http://confocal-microscopy-list.275.s1.nabble.com/timeseries-large-images-and-focus-tp5747682p5767682.html

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Hi Hank- PFS is not supposed to work with plastic.  Are you saying that you got it to work with a standard plastic multiwell plate or were you using a glass bottom dish?  Dave

On Nov 19, 2010, at 11:01 AM, Adams,Henry P wrote:

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> Sarah,
> I have a Ti with PFS and we do long timelapse experiments quite a bit with multiwell plates and also running 4 culture dishes.
> It is very important to make sure that your slide, plate or dishes are level as possible. I have had similar problems with PFS failing but by adjusting the set screws on the stage insert while using a small level tool on the plate or dishes I have been able to get PFS to work.
> Good luck,
> Hank Adams
> Microscopy Core
> Genetics
> U.T.M.D.Anderson Cancer Center
> Houston, Tx
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gabor Csucs
> Sent: Friday, November 19, 2010 5:03 AM
> To: [hidden email]
> Subject: Re: timeseries, large images and focus
>
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> Dear Sarah,
>
> I'd suggest that your PI invest in the Perfect Focus System (PFS) and
> that would solve these problems (given that you are using an inverted
> stage).
>
> Cheers   Gabor
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>> Hello,
>>
>> I am using a Nikon A1 confocal and I am trying to take a time series over several hours,  scanning across channels in a microfluidic chamber - my channels are 20 fields of view across, there are three channels.
>>
>> I am finding that even with one long (20 fields of view) image across one channel the focus is lost half way across, and moving to another region nearly always loses focus.
>>
>> I could increase the pinhole or take a small z stack so that being out of focus would matter less - I'm looking at bacteria ~1 micron in size, but don't need to resolve anything smaller, just distinguish between them using fluorescent labels.
>>
>> I wondered whether in principle this should work, using long image and PFS with several starting points, and if anyone has something similar working well, or whether I am expecting too much.
>>
>> Sarah
>> (student)
>
>
> --
> Gabor Csucs
> Light Microscopy Centre, ETH Zurich
> Schafmattstrasse 18, HPM F16
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> Web: www.lmc.ethz.ch
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Dr. David Knecht    
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)