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
>|
>|