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To all,
I should qualify my statement about phase problems using multiwell dishes. This is universal when using low mag objectives like the 10x/0.3 phase in the case I was describing.

hank

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Adams,Henry P
Sent: Tuesday, November 23, 2010 5:25 PM
To: [log in to unmask]
Subject: Re: time series, large images and focus

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Hi Dave,
I just tried PFS using a plastic 48-well plate and a 10x phase. I setup 5 positions with offsets in 4 different wells with water. It worked PERFECTly. I then rotated in my 20x/0.75. Five different positions within one well (similar to 35mm plastic dish) and it worked ok. Although, I am not sure what practical use the 20x would be for imaging.  However, PFS did not work when I attempted to setup positions in two adjacent wells. Also, this was not a phase contrast objective. I don't believe their 20x phase is compatible with their PFSystem. 
Also, phase contrast is tricky in multiwell dishes as most of you know. Unless you are near the center of the well, in my experience, it is impossible to get a phase contrast image.  Fyi, all my users who were using the 10x phase in multiwell dishes were conducting scratch assays. The images away from the center of wells made me cringe. But they claim to get what they needed. Ugh.

hank

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of David Knecht charter
Sent: Tuesday, November 23, 2010 12:12 PM
To: [log in to unmask]
Subject: Re: time series, large images and focus

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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:[log in to unmask]] On Behalf Of Gabor Csucs
> Sent: Friday, November 19, 2010 5:03 AM
> To: [log in to unmask]
> 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
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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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: +41 44 633 6221
> Mobile: +41 79 758 21 58
> Fax: +41 44 632 1298
> e-mail: [log in to unmask]

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)