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