On May 24, 12:03pm, Menczel Laszlo wrote:
>
> We would like to measure changes in the volume of cultured mammalian
> cells. The changes occur in about 10-20 seconds, and we would like to
> do some kinetics studies on them. So we need fairly fast sampling rate
> (like every second or half-second). We have a ZEISS LSM that can be
> used for the experiments.
 
 
Dear Menczel,
 
Just a few quick thoughts...
 
Since the changes are pretty fast you won't be able to do 3D reconstruction
during the change although control and final volume measurements should be
straightforward with a dextran-linked indicator in the extracellular fuid.
Perhaps you could get a water soluble indicator into the cell and record
changes in the concentration of that indicator to measure the cell water
volume? Such an indicator must cross membranes (i.e. leave and enter the cell)
only slowly so it might be necessary to "load" the cell with it for several
hours before enough gets in... Perhaps a medium weight dextran linked indicator
might be suitable. If this is possible, then the fluorescence reported by the
confocal should be proportional to the concentration of the indicator which is,
in turn, inversely proprtional to the volume of the cell (since you record from
a constant voxel).
A control experiment would be to compare the reported %change against full 3D
reconstruction once the reported signals are 'steady'.
 
I hope this helps..
 
Regards
Mark Cannell
 
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