Regarding the AM hypothesis, I wouldn't recommend it in fungal cells - I
am afraid it won't work. Some years ago I tried to use SBFI (a sodium
ratiometric dye) in marine fungal cells (Dendryphiella) and the dye would
not cleave properly. In diatom cells I could get some cleavage of
calcein, but the results were not that good, and with SBFI it was even
worse.  The only way to go with the AM dyes was to microinject the active
form.  I would also vote for eosin.

Good luck,

Grazyna Tokarczyk
Dept. of Psychology
Dalhousie Univ.
Halifax, Canada


On Thu, 2 Mar 2000, Jose Feijo wrote:

> Regarding NMR, perhaps it should be usefull to remember that this is NOT a single cell technique, rather a population measurement, usually starting with, at least, 1 gram of material. And assumes, of course, that all cells in a population behave reasonably synchrousnouly. Guess that rules out most system I know...
>
> Regarding the AM hypothesis, again that may be usefull for animal cells, but I doubt it will work reliably in plant/fungal cells, where there are esterases everywhere, including the cell wall, and where a significant proportion on unchanged dye goes directly to the vacuole and ER without de-esterification, creating an highly uneven distribuition of the dye, which I can't see easily how to discriminate in terms of kinetics. Even for most ratiometric dyes, AM-esters are simply useless in plants and fungi cells.
>
> Still, I would vote for eosin.
>
>
>
>
> *********** REPLY SEPARATOR  ***********
>
> On 01-03-2000 at 9:04 marshall montrose wrote:
>
> >Hello happy confocalists and Haixin Xu in particular,
> >
> >If your tisssues/cells will cleave acetoxymethyl esters, you can load
> >intracellularly with calcein/AM which will be converted to calcein in the
> >cytosol. In a confocal microscope (or in a pinch a regular microscope using
> >objective lense with high NA), the concentration of calcein can be
> >approximated by the cytosolic fluorescence.
> >
> >When water goes into the cells, they will swell and the calcein will be
> >diluted, and the fluorescence will go down. This is only a single wavelength
> >measurement, so there are lots of potential artifacts, but it does work.
> >-------
> >
> >Back to the magnetic resonance question, there IS a way to measure
> >intracellular water specifically. If you use proton NMR to measure the water
> >peak, you will certainly catch all water. However the weak intracellular
> >signal can be detected by its shift from the HUGE extracellular peak by
> >using the shift in mobility (I don't remember if it is T1 or T2 signal) for
> >the intracellular versus extracellular
> >
> >I don't know if the small intracellular signal will allow this to be
> >possible with NMR imaging, but it can be used with more conventional NMR
> >spectroscopy.
> >
> >Chip Montrose
> >
> >==========================================================
> >Marshall H. Montrose, Ph.D.
> > Indiana University                 tel: 317-278-3674
> >   Med Sci 307H                       fax: 317-278-3840
> >    635 Barnhill Drive                   e:  [log in to unmask]
> >     Indianapolis, IN 46202-5120
> >==========================================================
> >
> >
> >|  -----Original Message-----
> >|  From: Confocal Microscopy List
> >|  [mailto:[log in to unmask]]On Behalf Of haixin xu
> >|  Sent: Friday, February 25, 2000 1:49 PM
> >|  To: [log in to unmask]
> >|  Subject: water tracer dye?
> >|
> >|
> >|  Hi Gurus,
> >|  We want to know which part/cells of tissue takes water up first in a
> >|  fungal system. I am  wondering if somebody in this group knows some
> >|  techniques or some dyes (water tracer dye?) that could help us to do
> >|  this work?
> >|  I would very much appreciate any response.
> >|
> >|  Haixin Xu  Ph.D.
> >|  Department of Botany and Plant Pathology
> >|  Michigan State University
> >|  East Lansing, MI 48824
> >|
> >|
>