High temporal resolution video imaging of intracellular calcium

We have developed a system for imaging intracellular free calcium ion concentration ([Ca2+]i) at the highest rate possible with conventional video equipment. The system is intended to facilitate quantitative study of rapid changes in [Ca2+]i in cells that move. It utilizes intensified video cameras with nearly ideal properties and digital image processing to produce two images that can be ratioed without artifacts. Two dichroic mirrors direct images of cellular Indo-1 fluorescence at two different wavelengths to two synchronized video cameras, each consisting of a fast micro-channel plate image intensifier optically coupled with a tapered fiber optic bundle to a CCD image sensor. The critical technical issues in this dual-image system are: (1) minimization and correction of the small geometric and other types of differences in the images provided by the two cameras; and (2) the signal-to-noise ratio that can be achieved in single frames. We have used this system to obtain images of [Ca2+]i at 16.7 ms intervals in voltage-clamped single cardiac cells perfused internally with Indo-1 (pentapotassium salt). The images indicate that, except for the nuclear regions, [Ca2+]i is uniform during normal excitation-contraction coupling. In contrast, changes in [Ca2+]i propagate in rapid 'waves' during the spontaneous release of Ca2+ that accompanies certain 'Ca2(+)-overload conditions.'