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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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