Re: Oil objective maximum area/distance for long-term timelapse imaging

Posted by Smith, Benjamin E. on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Oil-objective-maximum-area-distance-for-long-term-timelapse-imaging-tp7584835p7584839.html

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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]> on behalf of Emmanuel Levy <[hidden email]>
Sent: Wednesday, March 2, 2016 8:46 AM
To: [hidden email]
Subject: Re: Oil objective maximum area/distance for long-term timelapse imaging

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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]> wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on 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]
> >
> 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: [phone]
> >
> > office: [phone] New number!
> >
> > LCI website
> >
> >
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of Kenneth Chen
> > Sent: den 2 mars 2016 13:22
> > To: [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 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
> (C) 508 507 1310
>