The issue of measuring subcellular calcium transients is a tricky
one that is steeped in controversy, particularly the issue of whether
a persistent calcium gradient can be maintained across the nuclear envelope.
One must take into account issues such as compartmentalization of indicator,
binding of indicator, spectral shifts and Kd shifts.  While imaging almost
always reveals nuclear/cytoplasmic fluorescence gradients or ratio gradients,
none of these has ever been proven to be a persistent calcium gradient
because of the enormous difficulty of subcellular calibration of
fluorescent calcium indicators.  Stephen Baylor has recent papers in the
Biophysical Journal on indicator behavior and my '94 J. Neurosci. paper
(14:5741) highlights the difficulties in calibration and reviews the older
literature.
    These problems seem equally applicable to subcellular cytoplasmic
fluorescent (calcium?) gradients.  As far as I know, when using fluorescent
calcium imaging, you can not resolve with even the best confocal microscope
(or 2 photon), the interstitial spaces between organelles from
the organelles themselves.  With confocal you should be able
to getter "closer" to the truth, but fluorescent indicators have many
devious tricks and one should interpret any persistent fluor/ratio gradients
very cautiously.

Nonetheless, this is still a lot of fun and there are tricks we
can use such as double labeling and looking at the calcium dynamics
to infer what is going on.

Don O'Malley
Dept. Biology
Northeastern University