Re: Protocol for imaging C. elegans on inverted spinning disk confocal microscope (industry response)

[Roy Edward-2]

Dear Jay

The thermo-reversible hydrogel mountant CyGEL has been used for this application.
Live C. elegans can be anaesthetized with Azide/Levamisole, as required, and then placed in cooled CyGEL which is then pipetted into the well/slide for imaging.  

Worms can be imaged like this for at least an hour.  You can see an example of this here:
http://www.biostatus.com/product/cygel/live_c_elegans_imaging/
which shows detail of embryogenesis from origin to zygote in one individual (GFP in the germline).

CyGEL is a liquid when cold and gels (reversibly) above 22 degC.  Gelling is firm and provides complete immobilization. This can be used in chambered slides, micro-plate wells or simply under a coverslip.  In wells, CyGEL can be overlaid with oil for oil immersion optics. CyGEL is easier to use than methyl cellulose, unlike LMP Agarose it does not cause hyper-thermal shock and is optically inert and clear with a RI equivalent to water. This makes it compatible with fluorescence microscopy and high-resolution techniques such as FRAP. (See Price et al. Molec.Biochem.Parasitol.169(1)66-69 for such use in live parasite imaging). Melting CyGEL by cooling allows recovery of the live objects by centrifugation/dilution.
(I have found an inexpensive and easy way to make wells on slides. If you want to know more about that please contact me off-list.)

A recent review on CyGEL is available in the current issue of American Biotechnology Laboratory or as a pdf here:
http://www.biostatus.com/resources/technical_documents/cygel_review_american_biotechnology_laboratory/

and on tumor-derived spheroids (Robertson et al, MD Anderson Cancer Center) in the current issue of J.Biomolec. Screen.:
http://jbx.sagepub.com/content/15/7/820

Full details on the technology can be found here:
http://www.biostatus.com/purchase/product.php?productid=18&cat=3&page=1

Don't hesitate to contact me if you have any questions.

Very best regards
Roy  
Roy Edward            
Biostatus Limited                    
Tel: [phone] Fax: [phone]
[hidden email] www.biostatus.com Company No: 3079239.
Registered in England and Wales. Registered office: Biostatus Ltd, 56 Charnwood Road, Shepshed, Leicestershire, LE12 9NP, UK.

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Date:    Wed, 25 Aug 2010 11:02:54 -0500
From:    Jay Vyas <[hidden email]>
Subject: Protocol for imaging C. elegans on inverted spinning disk confocal microscope

Hi-
We have a collaborator who is interested in confocal microscopy of C.elegans. We have not done work with this organism and are looking for the best way to immobilize the worm and prepare the sample for imaging on our spinning disk confocal microscope. We have a typical inverted microscope set-up (Nikon body).
The collaborator prefers to keep the worm(s) alive, so we need a method to immobilize them (anesthesia?).
Do folks use agar when mounting samples? Which slides are typically used for this purpose? If there is a reference, I would love to get the citation.

Thanks,
Jay Vyas
Massachusetts General Hospital