Re: time series, large images and focus

Posted by Sylvie Le Guyader-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/timeseries-large-images-and-focus-tp5747682p5755929.html

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Hi Hank

I suppose you use a dry objective for your multiwell experiments is that
right?

Med vänlig hälsning / Best regards
 
Sylvie
 
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Sylvie Le Guyader
Live Cell Imaging Unit
Dept of Biosciences and Nutrition
Karolinska Institutet
14183 Huddinge
Sweden
office: [phone]
This number will change to [phone] on the 13th of December 2010
LCI room: [phone]
mobile: [phone]
 

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of
> Adams,Henry P
> Sent: 19 November 2010 17:01
> To: [hidden email]
> Subject: Re: time series, large images and focus
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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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> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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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
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
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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
> CH-8093, Zurich, Switzerland
>
> Web: www.lmc.ethz.ch
> Phone: [phone]
> Mobile: [phone]
> Fax: [phone]
> e-mail: [hidden email]