John J. Lemasters wrote:

> Because confocal microscopy collects a optical slice of uniform thickness, the
> importance of ratiometric measurement is diminished since the fluorescence
> intensity becomes mostly independent of cell shape and volume. Ratioing
> helps to calibrate concentration but there are also other ways to do this
> without ratioing.

Sorry to pull us off the current thread, but I had to take issue with
this statement (sorry John). I think ratioing is at least as crucial in
confocal/two-photon microscopy as in conventional microscopy. I heartily
agree that we are liberated from cell shape and volume effects because
of sampling from a subcellular volume, BUT there is also a price.

Optical sectioning makes measurements exquisitely sensitive to changes
in focus and position (and we are discussing live cells here). Depending
on the system, laser scanning can make it more difficult to avoid
photobleaching. Finally if you have any interest in long term events,
you will have to worry about dye leakage. I use emission ratio imaging
as my security blanket against all these problems.

I agree with John that one can work around some/all of these problems,
but I don't think it should be a first line choice if ratioing is
possible.

Chip
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M.H. Montrose, Ph.D.
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