http://confocal-microscopy-list.275.s1.nabble.com/Oil-objective-maximum-area-distance-for-long-term-timelapse-imaging-tp7584835p7584847.html
Thanks for your detailed answer. This problem means we have avoided using
We generally cover the full plate with oil and image it. In terms of
wells for ~24h and it's been working well. Like everyone described, it is
important to slow down stage movements. Also, as I mentioned in a previous
post, the viscosity of the oil matters. The one we use now is from
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
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> *****
>
> Hi Emmanuel-
>
> 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.
>
> -Jason
>
>
> On 2 March 2016 at 19:19, Miller, Jason <
[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/> and include
> the link in your posting.
> > *****
> >
> > 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: *
[hidden email]<mailto:
>
[hidden email]><mailto:
> >
[hidden email]<mailto:
[hidden email]>>
> <
[hidden email]<mailto:
[hidden email]
> ><mailto:
> >
[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:
>
[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]>>>
> 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]
> >>
> > > 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:
>
[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_Offers> >
> > > > > 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]
> >>]
> > > > > On Behalf Of Kenneth Chen
> > > > > Sent: den 2 mars 2016 13:22
> > > > > To:
[hidden email]<mailto:
>
[hidden email]><mailto:
> >
[hidden email]<mailto:
[hidden email]
> >>
> > > > > 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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> >
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