Re: time series, large images and focus

Posted by Adams,Henry P on
URL: http://confocal-microscopy-list.275.s1.nabble.com/timeseries-large-images-and-focus-tp5747682p5755870.html

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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
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CH-8093, Zurich, Switzerland

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