Fluorescent staining of living S. Pombe

[lgelman]

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Hello everybody,


We would need to stain living S. Pombe cells imaged on a fluorescence microscope to segment thereafter during image analysis automatically the cells.
Staining cell wall and/or membrane would be perfect. Has someone any specific experience with yeast and/or Pombe?

Note that we need an organic dye and cannot express any recombinant protein (we are screening a yeast library).

Thanks for your advices!

Best regards,


Laurent.



_____________________________________________
Laurent Gelman, PhD
Head of Facility for Advanced Imaging and Microscopy
(Light Microscopy)

Friedrich Miescher Institut
WRO 1066.2.16
Maulbeerstrasse 66
CH-4058 Basel
[phone]
[phone]

[rjpalmer]

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Laurent -
Calcofluor or other cellulose-binders are typical, caveat being I  
don't work with yeasts..  A membrane stain called FUN has also been  
used.  See papers with Ghannoum in the author line.
Rob

On Aug 15, 2013, at 5:46 AM, Gelman, Laurent wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello everybody,
>
>
> We would need to stain living S. Pombe cells imaged on a  
> fluorescence microscope to segment thereafter during image analysis  
> automatically the cells.
> Staining cell wall and/or membrane would be perfect. Has someone any  
> specific experience with yeast and/or Pombe?
>
> Note that we need an organic dye and cannot express any recombinant  
> protein (we are screening a yeast library).
>
> Thanks for your advices!
>
> Best regards,
>
>
> Laurent.
>
>
>
> _____________________________________________
> Laurent Gelman, PhD
> Head of Facility for Advanced Imaging and Microscopy
> (Light Microscopy)
>
> Friedrich Miescher Institut
> WRO 1066.2.16
> Maulbeerstrasse 66
> CH-4058 Basel
> [phone]
> [phone]

Robert J. Palmer Jr., Ph.D.
Microbial Receptors Section
Laboratory of Cell and Developmental Biology
Natl Inst Dental Craniofacial Res - Natl Insts Health
Bldg 30, Room 207
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax [phone]

[Graham Wright-2]

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Hi Laurent,

How about FM4-46 (it will stain the membranes, initially the plasma membrane)? One potential problem though, depending in the rate of endocytosis in your cells, will be that it will be internalised over time if the cells will be in it for a while before imaging. It worked well with Neurospora crassa for us.

Regards
Graham

--
Graham Wright, PhD
Institute of Medical Biology, A*STAR
Singapore

On 15 Aug, 2013, at 20:50, "Rob Palmer" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Laurent -
> Calcofluor or other cellulose-binders are typical, caveat being I  
> don't work with yeasts..  A membrane stain called FUN has also been  
> used.  See papers with Ghannoum in the author line.
> Rob
>
> On Aug 15, 2013, at 5:46 AM, Gelman, Laurent wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello everybody,
>>
>>
>> We would need to stain living S. Pombe cells imaged on a  
>> fluorescence microscope to segment thereafter during image analysis  
>> automatically the cells.
>> Staining cell wall and/or membrane would be perfect. Has someone any  
>> specific experience with yeast and/or Pombe?
>>
>> Note that we need an organic dye and cannot express any recombinant  
>> protein (we are screening a yeast library).
>>
>> Thanks for your advices!
>>
>> Best regards,
>>
>>
>> Laurent.
>>
>>
>>
>> _____________________________________________
>> Laurent Gelman, PhD
>> Head of Facility for Advanced Imaging and Microscopy
>> (Light Microscopy)
>>
>> Friedrich Miescher Institut
>> WRO 1066.2.16
>> Maulbeerstrasse 66
>> CH-4058 Basel
>> [phone]
>> [phone]
>
> Robert J. Palmer Jr., Ph.D.
> Microbial Receptors Section
> Laboratory of Cell and Developmental Biology
> Natl Inst Dental Craniofacial Res - Natl Insts Health
> Bldg 30, Room 207
> 30 Convent Drive
> Bethesda MD 20892
> ph 301-594-0025
> fax [phone]

[Theresa Swayne]

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Dear Laurent,

Fluorescent Concanavalin A and Calcofluor both work well for S. cerevisiae.  

Calcofluor is the cell wall dye used in the FUN-1 (Live/Dead) yeast kit.

The Ohya and Morishita labs used FITC-ConA to generate the Saccharomyces cerevisiae Morphological Database, http://scmd.gi.k.u-tokyo.ac.jp/datamine/.

If you are doing long-term imaging, ConA might be better as you could choose wavelengths that would avoid UV exposure.

Hope this helps!

Theresa



On Aug 15, 2013, at 5:46 AM, "Gelman, Laurent" <[hidden email]> wrote:

>
> We would need to stain living S. Pombe cells imaged on a fluorescence microscope to segment thereafter during image analysis automatically the cells.
> Staining cell wall and/or membrane would be perfect. Has someone any specific experience with yeast and/or Pombe?
>
> Note that we need an organic dye and cannot express any recombinant protein (we are screening a yeast library).
>
>

------------------------------------
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/research/sharedresources/confocal