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Hi Michael,
Could it be because you cells are moving a different amount laterally to
your coverslip? PFS (and Olympus ZDC) just find the coverslip again each
time. They assume your sample stays at the same lateral position in
relation to the coverslip. So if you cover slip moves laterally 10um but
your cells move 20um the PFS will only compensate for the 10um drift and
your cells will still be out of focus.
Cheers
Cam
Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research Melbourne - Parkville Branch
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104
Facility Website
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Cammer, Michael
Sent: Friday, 22 July 2011 12:55 AM
To: [log in to unmask]
Subject: Re: Olympus ZDC 2 vs. Nikon PFS
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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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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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