*****
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 Radek,

In an exact set up as yours, I had encountered similar problems. To
overcome the issues I did the following
- Before putting the multiwell chamber on to the stage, I made sure that
the microscope was well equilibrated with the incubation chamber at 37C
(please see the note toward the end)
- The bottom part of the glass coverslip was coated with film of oil by
rolling the cylinder part of the glass tip applicator. In my experience,
using the glass tip applicator reduced the bubbles
- Oil was put on the objective and the plate was set on the stage
- A contact was made between the objective and the plate and the cells were
brought in focus
- The plate was allowed to equilibrate at 37C for 30 minutes
- All the region of interests on the multiwell plate were selected in the
software and the perfect focus (reflection based focus value) set for each
point. Because of slight slant, as I moved from well to well the focus
changed. Not waiting for 30 minutes to equilibrate also resulted in
incorrect reflect based focus values for the entirety of the experiment
- The plate was made to travel through this path manually a couple of times
to make sure that oil is evenly spread out and there is no possibility of a
bubble forming later due to an insufficient oil

Note: Initially, my experiments failed because the thermal drift with only
the stage top incubator was overwhelming for a reflection based system to
manage after a few hours. Using both the stage top incubator and an
incubator that encloses an entire microscope, I have done time-lapse for
upto 20 hours.

Happy to elaborate or clarify any of these points.

Best,
Gaurav.

*Gaurav Joshi, PhD*  |  Imaging Scientist
Health Sciences Research Building (HSRB)
EG72, 1760 Haygood Drive, Atlanta, GA 30322

[log in to unmask]
ici.emory.edu <http://www.cores.emory.edu/ici/>



On Sun, Jun 21, 2020 at 2:57 AM Radek Machan (Dr) <[log in to unmask]>
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 and include the link in your posting.
> *****
>
> Dear All,
>
> One of our users is doing long-term time-lapse imaging in multi-well
> plates with reflection based autofocus. The sample is quite flat, so an oil
> immersion lens seemed the best choice in terms of light collection
> efficiency and image quality. Our first run worked perfectly well. However,
> during the second experiment the focusing failed quite early during the
> time-lapse and the sample remained out of focus for most of the experiment.
> I suspect that an air bubble in the oil immersion could have interfered
> with the focusing and because of high viscosity of the oil, a bubble can
> persist there for quite some time. I'm wondering now, if the hypothesis of
> the bubble is correct, how likely is such an incident to happen - whether
> we were particularly lucky in the first experiment or particularly unlucky
> in the second.
>
> What is your experience with the use of oil immersion in such experiments?
>
> Thanks! Best wishes,
> Radek
>
> Radek MACHAN, Ph.D. (Senior Research Fellow)
> SCELSE Advanced Biofilm Imaging Facility<
> http://www.scelse.sg/Page/imaging-facility> Manager
> Nanyang Technological University
> #B2, 60 Nanyang Drive, SBS-01N-27
> Singapore 637551
>
>
> ________________________________
>
> CONFIDENTIALITY: This email is intended solely for the person(s) named and
> may be confidential and/or privileged. If you are not the intended
> recipient, please delete it, notify us and do not copy, use, or disclose
> its contents.
> Towards a sustainable earth: Print only when necessary. Thank you.
>