Live cell imaging prep. for yeast S. pombe

[Graham Wright]

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

[mahogny]

Graham Wright 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
>  

for c.elegans embryos we use polylysine to glue them down. to make
sure they aren't crushed, we add ~10 50um beads.

polylysine seems to work best if you add a droplet and then lets it dry
on a heat block, probably due to some crystallization.

/Johan

--
--
------------------------------------------------
Johan Henriksson
MSc Engineering
PhD student, Karolinska Institutet
http://mahogny.areta.org http://www.endrov.net

[Jörg Bormann]

Hello,
for live-cell imaging of (filamentous) fungi we use agarose instead of agar. This diminishes autofluorescence to some extent. Polylysine didn´t work that well with filam. fungi.
Best wishes - Joerg

_____________________

Jörg Bormann
Westfälische Wilhelms-Universität Münster
Institut für Botanik
Schlossgarten 3
48149 Münster
Phone: [phone]

2009/5/22 Graham Wright <[hidden email]>

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

--

[Theresa Swayne]

Graham, 

My experience is with cerevisiae, but perhaps it will help. First, we mix agarose with synthetic medium (aka SC).  It's less autofluorescent than our yeast extract-based medium (YPD). Second, we use a thinner agarose layer. Our agarose pad is < 100 um thick  but the cells are fine for hours. 

In our hands, a simple glass-slide mount does not support normal cell physiology for very long (~15 min).

For details, feel free to e-mail me off list, or see 

Visualization of mitochondria in budding yeast.

Swayne TC, Gay AC, Pon LA.

Methods Cell Biol. 2007;80:591-626.

PMID: 17445715 

Good luck,

Theresa

On May 22, 2009, at 2:01 AM, Graham Wright wrote:

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. 

----------------------------------

Theresa C. Swayne, Ph.D.

Manager, Confocal and Specialized Microscopy Shared Resource

Herbert Irving Comprehensive Cancer Center, Columbia University

1130 Saint Nicholas Ave, 222A

New York, NY 10032

212-851-4613

[hidden email]

http://hiccc.columbia.edu/?page=research/sharedresources/confocal

[Bruno Afonso-2]

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)