http://confocal-microscopy-list.275.s1.nabble.com/Oil-objective-maximum-area-distance-for-long-term-timelapse-imaging-tp7584835p7584849.html
We have seen this problem before on our Nikon systems but this seem to have been a software problem so it was fixed by an update. We are currently running on NIS 4.40 and do not experience this anymore either with the 20x air or the 60x oil objectives. You might want to ask Nikon for a hot fix in case you are running on an older version.
Subject: Re: Oil objective maximum area/distance for long-term timelapse imaging
I thought I was the only one who had noticed this problem! I've not seen it discussed on the listserve, and when I contacted Nikon, they hadn't heard of the problem. But it soon became clear to me that the issue you bring up is exactly what's going on. Here's what I've noticed about the phenomenon and some things I've done to avoid the problem. We use the Nikon PFS system, which as you know, relies on an IR beam reflecting off a refractive index mismatched surface. We see the problem manifest as a "jitter" as PFS rapidly jumps between planes (the objective water-glass interface, and then the glass surface-cell interface).
a) If you have confluent cells, especially if they are densely packed, that decreases the problem since the RI of the cells is different than pure water.
b) The higher the NA objective, the worse the problem. Although to be honest, I haven't distinguished whether it's higher objective magnification or higher NA specifically.
c) The thinner the glass, the worse the problem. If you are using an objective with a correction collar, that means you can try for glass that is thicker and adjust by adjusting the correction collar.
Our main solution for automated imaging involved writing a micromanager script. What we found out was that if perfect focus starts to jitter at a spot (jumping between the top and bottom of the glass), we let PFS go for a few seconds and take the average Z position of where it is during the jitter (get Z position every x ms, and average that position over the few seconds we let PFS go). Then we turn PFS off, have the stage move to this Z position (the average Z depth), and turn PFS back on. 95% of the time, that's all we need. Occasionally, that doesn't work. If it doesn't, we try one more time. And then if that doesn't work, we assume we are instead out of PFS range, and we have the script automatically move X um up, then X um down from the original Z focus spot and reattempt PFS focus there. The X um is determined empirically, but once you determine it, it's remarkably reproducible. We've been able to do runs over entire plates for 40+ hours, imaging every 20 minutes to 1 hour, using this type of setup.
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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> *****
>
> We tried to solve this problem by using water instead of oil,
> acknowledging that we lost the increased NA. We were using an inverted
> setup, and similar to Sylvie, we machined a cap that goes around the
> objective with a drip setup - so water drips into this cap, which
> encases the objective, and keeps water in the cap at all times. You
> have to empirically test what drip rate you need to counter
> evaporation. We found that the viscous water substitutes (e.g.
> ultrasound gel, gonisol, etc.) did not have enough capillary action to
> stay sealed between the objective and the bottom of the plate. But
> water did. This also allows for very fast imaging - you don't have to
> slow down the speed with which the stage travels between wells or
> within wells at all.
>
> Regards-
> Jason
>
> -------
>
> Jason Miller, MD, PhD
>
> University of Michigan Kellogg Eye Center
>
>
> Home address:
>
> 117 Worden Ave
>
> Ann Arbor, MI 48103
>
> Cell: (415)
> 225-2134<tel:%28415%29%20225-2134><tel:%28415%29%20225-2134>
>
> E-mail: *
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>
[hidden email]<mailto:
[hidden email]>> <
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>
[hidden email]<mailto:
[hidden email]
> >>>*
>
>
> "Forgiveness does not change the past, but it does enlarge the
> future." - Paul Boese
>
> On Wed, Mar 2, 2016 at 12:05 PM, Sylvie Le Guyader <
>
[hidden email]<mailto:
[hidden email]><mailto:Sylvie.
>
[hidden email]<mailto:
[hidden email]>>>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > Post images on
> >
http://www.imgur.com<
http://www.imgur.com/><
http://www.imgur.com/>
> > and include
> the link in your posting.
> > *****
> >
> > I forgot to write that what the magic oil device does is that it
> > refills the immersion oil on the objectuf lense. :-)
> >
> > I agree that reducing the stage speed helps but we never got to a
> > fully reliable system. Might take magic hands... Sigh!
> >
> > Ann. What do you mean by 'it did not keep'? Did it bleach? The
> > GattaQuant website specifically says that it virtually doesn't
> > bleach! :-(
> >
> > Med vänlig hälsning / Best regards
> >
> > Sylvie
> >
> > @@@@@@@@@@@@@@@@@@@@@@@@
> >
> > Sylvie Le Guyader, PhD
> > Live Cell Imaging Unit Manager
> > Karolinska Institutet- Bionut Dpt
> > Hälsovägen 7,
> > Novum, G lift, floor 6
> > 14157 Huddinge
> > Sweden
> > mobile: +46 (0) 73 733
> > 5008<tel:%2B46%20%280%29%2073%20733%205008><tel:%2B46%20%280%29%2073
> > %20733%205008>
> > office: +46 (0) 8 5248 1107<tel:%2B46%20%280%29%208%205248%201107>
> > LCI website
> >
> >
> > ---- Smith, Benjamin E. wrote ----
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > Post images on
> >
http://www.imgur.com<
http://www.imgur.com/><
http://www.imgur.com/>
> > and include
> the link in your posting.
> > *****
> >
> > I can also testify to the benefit of greatly reducing the stage
> > velocity during long-term live imaging. We have done imaging of up
> > to 48-hours, and having the stage crawl along allows the immersion
> > fluid to stay
> wetted
> > to the objective, and also eliminates any sample motion.
> >
> > One other consideration we've done is to setup the run early in the
> > morning so you can check up on it throughout the day. That way, you
> > can also add immersion fluid as needed. Normally, you will find
> > that towards the beginning of the run you may need to add a little
> > more to the objective, but after about 3-4 hours everything
> > stabilizes, and the
> system
> > is good to go.
> >
> > I also make sure that there is a sufficient pause between scans so
> > that I have enough time to adjust things and still start the next
> > scan at
> exactly
> > the next time interval.
> >
> > Good luck,
> > Ben Smith
> >
> > ________________________________________
> > From: Confocal Microscopy List <
[hidden email]<mailto:
[hidden email]><mailto:
>
[hidden email]<mailto:
[hidden email]
> DU>>> on
> > behalf of Emmanuel Levy <
[hidden email]<mailto:
[hidden email]><mailto:
>
[hidden email]<mailto:
[hidden email]>>>
> > Sent: Wednesday, March 2, 2016 8:46 AM
> > To:
[hidden email]<mailto:
[hidden email]><mailto:
>
[hidden email]<mailto:
[hidden email]
> DU>>
> > Subject: Re: Oil objective maximum area/distance for long-term
> > timelapse imaging
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > Post images on
> >
http://www.imgur.com<
http://www.imgur.com/><
http://www.imgur.com/>
> > and include
> the link in your posting.
> > *****
> >
> > It may also help to use oil with high viscosity index so it remains
> spread
> > on the glass.
> > All the best,
> > Emmanuel
> >
> >
> > On 2 March 2016 at 16:18, Gary Laevsky <
[hidden email]<mailto:
[hidden email]><mailto:
>
[hidden email]<mailto:
[hidden email]>>> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > > Post images on
> > >
http://www.imgur.com<
http://www.imgur.com/><
http://www.imgur.com/>
> > > and
> include the link in your
> > posting.
> > > *****
> > >
> > > We regularly image the 4 center wells (about 50 total points) of a
> > Labtek 8
> > > well chambered coverslip for 8-16 hours at 37c using PFS on a TiE.
> > >
> > > First we put the plate on, one drop of oil on the 60X or 100X
> objective,
> > > and drive to all four wells.
> > >
> > > Then we take the plate off, and add one more drop of oil, and
> > > we're
> good
> > to
> > > go.
> > >
> > > We also slow the stage speed way down so PFS can follow the glass.
> > >
> > > Hope this helps.
> > >
> > > Gary
> > >
> > > On Wed, Mar 2, 2016 at 7:49 AM, Sylvie Le Guyader
> > <
[hidden email]<mailto:
[hidden email]><mailto:Sylv
> >
[hidden email]<mailto:
[hidden email]>>
> > > >
> > > wrote:
> > >
> > > > Hi Ken
> > > >
> > > >
> > > >
> > > > My experience: imaging 2 cells 1.7mm apart (thickness of the
> > > > well
> wall
> > in
> > > > the MW plates we were using then) every 10 min for 12h with a
> > > > 60x oil objective and a hardware autofocus at 37 deg resulted in
> > > > a high risk
> > > (about
> > > > 25% of the cases?) of losing focus. There go years of sweat and
> tears...
> > > >
> > > >
> > > >
> > > > We were lucky to get to test a super device manufactured by
> > > > EMBLEM,
> > the
> > > > commercial side of EMBL. You send your objective. They custom
> > > > make a
> > cap
> > > > and send you a kit containing cap, tubes, pump and instructions.
> > > > It
> is
> > a
> > > > bit clumsy because you need to remove all other objectives and
> > > > you
> > need
> > > to
> > > > be careful not to put oil all over the place but it works a
> > > > charm. I
> > have
> > > > imaged 10 positions in each well over 15 wells every 10 min for
> > > > 15 h
> > with
> > > > this device without losing focus. :) You could contact them<
> > > >
> >
http://www.embl-em.de/articles.php?lang=de&hid=4&Cat=Technology_Offe> > rs>
> > > > to ask if they commercialize it.
> > > >
> > > >
> > > >
> > > > Med vänlig hälsning / Best regards
> > > >
> > > >
> > > >
> > > > Sylvie
> > > >
> > > >
> > > >
> > > > @@@@@@@@@@@@@@@@@@@@@@@@
> > > >
> > > > Sylvie Le Guyader, PhD
> > > >
> > > > Live Cell Imaging Facility Manager
> > > >
> > > > Karolinska Institutet- Bionut Dpt
> > > >
> > > > Hälsovägen 7,
> > > >
> > > > Novum, G lift, floor 6
> > > >
> > > > 14157 Huddinge
> > > >
> > > > Sweden
> > > >
> > > > mobile: +46 (0) 73 733
> > > > 5008<tel:%2B46%20%280%29%2073%20733%205008><tel:%2B46%20%280%29%
> > > > 2073%20733%205008>
> > > >
> > > > office: +46 (0) 08-524 811 72 New number!
> > > >
> > > > LCI website
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > -----Original Message-----
> > > > From: Confocal Microscopy List
> > [mailto:
[hidden email]<mailto:
[hidden email]><mailto:
>
[hidden email]<mailto:
[hidden email]
> DU>>]
> > > > On Behalf Of Kenneth Chen
> > > > Sent: den 2 mars 2016 13:22
> > > > To:
[hidden email]<mailto:
[hidden email]><mailto:
>
[hidden email]<mailto:
[hidden email]
> DU>>
> > > > Subject: Oil objective maximum area/distance for long-term
> > > > timelapse imaging
> > > >
> > > >
> > > >
> > > > *****
> > > >
> > > > To join, leave or search the confocal microscopy listserv, go to:
> > > >
> > > >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > > >
> > > > Post images on
> > > >
http://www.imgur.com<
http://www.imgur.com/><
http://www.imgur.com> > > > /> and
> include the link in your
> > > posting.
> > > >
> > > > *****
> > > >
> > > >
> > > >
> > > > Hi all,
> > > >
> > > >
> > > >
> > > > We're in the process of designing microfluidic devices for
> > > > multi-day time-lapse imaging. In the past we've had some trouble
> > > > with image
> > quality
> > > > for very large devices deteriorating due to the loss/thinness of
> > > > the immersion oil layer between sample and objective when very
> > > > large
> areas
> > > are
> > > > scanned. We have only used traditional oil objectives (no water
> > > > or
> > > >
> > > > glycerol) Does anyone have any strategies to mitigate this or
> > > > rules
> of
> > > > thumb about "safe" maximum sampling areas or distances to travel?
> > > >
> > > >
> > > >
> > > > Thanks,
> > > >
> > > > Ken Chen
> > > >
> > >
> > >
> > >
> > > --
> > > Best,
> > >
> > > Gary Laevsky, Ph.D.
> > > Confocal Imaging Facility Manager
> > > Dept. of Molecular Biology
> > > Washington Rd.
> > > Princeton University
> > > Princeton, New Jersey, 08544-1014
> > > (O) 609 258 5432<tel:609%20258%205432><tel:609%20258%205432>
> > > (C) 508 507 1310<tel:508%20507%201310><tel:508%20507%201310>
> > >
> >
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