Re: Live cell imaging using a inverted confocal microscope **Tech Support Response**

[Roy Edward-2]

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Dear Faizan,

You might want to have a look at this paper just published (on May 11th)
from researchers at the Univ of Utah and Cardiff Univ, UK:

Colombo, J. S., et al (2015). A 3D ex vivo mandible slice system for
longitudinal culturing of transplanted dental pulp progenitor cells.
Cytometry Part A.
doi: 10.1002/cyto.a.22680

http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22680/abstract

In this novel application, imaging of live mandible slices (ex vivo
micro-environments for study of GFP+ pulp progenitor cells) was performed
periodically using the thermo-reversible hydrogel mountant CyGEL
(PBS-based) to immobilise the object. This dealt with its irregularity
under a coverslip and thereafter permitted its non-destructive recovery
into its original medium for onward culture.  At the very least this will
suspend your tissue sample securely on the optical surface without
glueing, etc.

Similar methods have been applied to Danio embryos and drosophila larvae
for example and it seems appropriate for your application from what you¹ve
said.  

If specific nutrients are required during the imaging, one can use the
related CyGEL Sustain which is designed to accept 10X culture medium
specific to the cells or tissue (in your case) of interest.

One can create chambers out of silicone rubber o-rings - perhaps with 10
mm i.d. (given an estimated volume of 30-40 cu.mm at E12) and of
appropriate height to submerge into the CyGEL mountant.  These can be
smeared with silicone grease to create a waterproof seal with the slide.
I¹ve used thin (H = 1.5 mm) ones of these successfully for a number of
different applications. Alternatively, one could use a chambered slide.

CyGEL transits from sol to gel above room temperature (ca. 22 degC) and
remains a gel above 37 degC.  Chilling below 22 degC re-liquefies CyGEL
for re-positioning or non-destructive recovery (which might also be
achieved with excess volumes of cooled medium). Thus, this behaviour is
the opposite to that of LMP agarose.

A review of the technology can be found here:
American Biotechnology Laboratory. July/August issue: p12-15
http://www.nxtbook.com/nxtbooks/isc/abl_20100708/index.php?startid=12


You can get full technical details here:
http://www.biostatus.com/CyGel or http://www.biostatus.com/CyGEL-Sustain

Please feel free to contact me off-list if you would like to discuss this
in more detail.

Kind regards,
Roy

Roy Edward
E roy(at)biostatus(dot)com

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Date:    Wed, 27 May 2015 17:11:36 -0500
From:    Faizan Malik <[hidden email]>
Subject: Live cell imaging using a inverted confocal microscope

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Hi Everyone,

I am currently working on optimizing a protocol for live cell imaging of
embryonic brains (mice) with a inverted confocal microscope.

Does anyone have a specific set up or protocol they use? Or a certain type
of perfusion chamber design? I have found a lot of literature regarding
live cell imaging with normal confocal microscopes, but there does not
seem to be much there on live imaging with inverted confocal microscopes.
Is there any specific way that you attach your samples to the chamber in a
way that prevents damage to morphology as well as prevent reduced image
quality due to the using substances like glue or collagen to hold the
specimen in place (a problem not seen with non-inverted confocal
microscopes). We use a Zeiss LSM 700 in our lab.
Your help is appreciated!
Cheers,
Faizan