Re: Live cell imaging prep. for yeast S. pombe

Posted by Bruno Afonso-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Live-cell-imaging-prep-for-yeast-S-pombe-tp2955849p2957625.html

Hi,

Agarose is a great solution. Depending on how long you want to keep them growing in the scope you can play with how thick the agarose slab is. Make the slab out of the media you use. 2% is a good starting point. You can also use low-melt agarose. (more pillowy for the cells?)

Also, why don't you use deconvolution to get crisper images? People often forget that they can deconvolve wide-field microscopy and are often amazed by the results...

You can also use the concavilin-A method but it "wears off" with new generations. Agarose is more stable XYZ wise.

b

On Fri, May 22, 2009 at 02:01, Graham Wright <[hidden email]> wrote:

Hello,

We are doing some live cell imaging of Schizosaccharomyces pombe
(fission yeast). Usually the cells are mounted on an agar plug (agar &
YES media) on a concave slide, covered with a coverslip and sealed.
This works very well for confocal microscopy, but recently upon trying
widefield microscopy we have noticed a significant background
fluorescence, presumably from the agar. Simply mounting the cells in
liquid media on a normal glass slide significantly removes the
background problem, but creates another - the cells do not adhere to
the glass and so make long term timelapse imaging very difficult.

So, can anyone suggest ways of encouraging the cells to adhere that
work well for yeast, particularly S. pombe? Alternatively any
suggestions of agar, or other gelling agents that do not fluoresce
would be appreciated.

Thanks in advance for your help,
Graham

--
Bruno Afonso
http://brunoafonso.com (personal, mostly portuguese)
http://openwetware.org/wiki/User:BrunoAfonso (Professional, english)