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For what it's worth, our lab has used two PFS-enabled Nikon scopes for daily multi-XY time-lapse imaging for a couple of years now.  We did not compare PFS with any other system as competing focus control schemes were still largely software-based (and thus less appealing) in 2008.  In our experience lateral drift is almost always caused by the imaging chamber shifting in its mount.  One of our scopes has a spring-loaded chamber holder (in a piezo stage insert) and never experiences lateral drift.  The other scope has a different holder and had issues with XY drif until we glued some strips of rubber band to the sides of the chamber holder to prevent slippage.  That worked until the rubber band strips saturated with immersion oil and had to be changed. 

We almost always use 25mm coverslips in Attofluor chambers (Invitrogen), or else Mattek dishes with #1.5 glass.  In our experience PFS lock is easy and fast to acquire after a little practice.  The main impediment is normally small bubbles in the immersion oil.  Since using PFS I have become much more conscious of these - some days I will oil, clean and re-oil an objective once or twice to verify that the tiny bubbles are completely gone.  These can be the most frustrating because acquisition and imaging will work fine but PFS will randomly disengage during imaging as the bubble shifts around.  The other major reasons we lose focus are glass tilt, as others have mentioned, and evaporation of the cell medium.   Reasonable temperature shifts do not affect us much.  

Since glass tilt can be hard to notice, we only ask the PFS to make smallish jumps between each XY position.  If necessary we add an 'empty' XY position to break one large jump into two small ones.  The glass moves much faster while scanning for cells that it does during imaging so a problem can (frustratingly) only appear after you set everything up and press 'run'.   A little foresight prevents that.  

Dirt would certainly be a problem, but I am rather OCD about keeping glass clean.   

I hope this helps, 


TF

Timothy Feinstein, PhD
Postdoctoral Fellow 
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine 
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Jul 21, 2011, at 10:54 AM, Cammer, Michael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> Just wanted to give an update.
> 
> We did extensive tests with Tetraspeck beads imaged by TIRF and the Nikon PFS with the 100X TIRF objective held the focus for hours under different temperature conditions.  There was lateral drift of up to 10 um when we turned the heat on and off to check for focus locking during temperature shifts, but the focus held.  We were very happy about this.
> 
> However, when we switched to cells on lipid substrates in the exact same chamber, the focus did not hold.
> 
> So we are somewhat mystified and continuing to run tests.
> 
> Regards,
> 
> Michael
> 
> From: Craig Brideau [mailto:[log in to unmask]]
> Sent: Monday, July 18, 2011 12:34 AM
> To: Cammer, Michael
> Subject: Re: Olympus ZDC 2 vs. Nikon PFS
> 
> We have a Nikon PFS that mostly works.  We learned a few things early on that help make it more reliable.  It needs #1.5 cover slips.  If you use #1 it will overfocus as it is expecting #1.5 thick coverslips.  Dirt can be a big issue; the lens has to be fairly clean.  Sometimes dirt on the coverslip can throw things off as well.  Keeping the sample level is important, if there is any tilt it gets thrown off.  Finding the initial focus to get it to 'lock on' can require some patience occasionally.  You have to manually adjust the focus and keep trying to engage the PFS system.  It will eventually find the surface but it sometimes takes a few tries.  Usually it will get it on the first try though.  The performance is different for different objectives, so try all the PFS compatible objectives you have to see how they work.
> I hope these tips help!
> 
> Craig
> 
> 
> On Sat, Jul 16, 2011 at 6:16 PM, Cammer, Michael <[log in to unmask]<mailto:[log in to unmask]>> wrote:
> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> If there is anyone out there who has had problems with the Nikon PFS system drifting out of focus during experiments or problems with multiple field focus fidelity, would you please contact me privately.  We are trying to assess how common this problem is and, more importantly, want to know any tips to counter these issues.
> 
> Thank you!
> 
> _________________________________________
> Michael Cammer, Assistant Research Scientist
> Skirball Institute of Biomolecular Medicine
> Lab: (212) 263-3208  Cell: (914) 309-3270
> 
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