A first generation digital imaging video microscopy system has been developed that is able to provide both quantitative and visual three dimensional information from individual vascular endothelial cells. More specifically, with the combination of optical sectioning, video microscopy, digital image processing and analysis, deconvolution, fluorescence ratio imaging, and scientific visualization we are able to measure the dynamic changes in spatial distributions of intracellular ions. The technique presented involves the following steps: acquiring three dimensional biological data by optical sectioning of a specimen, preprocessing the digitized volume data, experimentally determining the digital imaging system’s point spread function (PSF), deconvolving the volume data with the PSF to remove the inherent out‐of‐focus information, and finally postprocessing and volume rendering in order to display the volume data in a quantitative and comprehensible manner. This technique permits high resolution visualization and quantification of three dimensional spatial distributions of ions as well as traditional temporal changes in ion concentration. It has the potential to aid immensely in research since the three dimensional spatial information is often a prerequisite for understanding the molecular mechanisms involved in many complex cellular processes. ©1995 American Institute of Physics.