Fluorescence microscopy of live cells is an important tool to investigate cellular tracking pathways. The existing microscope design is very well suited to image fast moving vesicles, tubules and organelles in one focal plane. More problematic is the imaging of cellular components that move between different focal planes. This is due to the fact that tracking of such cellular components requires that the focal plane of the microscope be changed. This has to be done with a focusing device, which is relatively slow. More importantly, only one focal plane can be imaged at a time. Therefore, while the cell is imaged at one focal plane, important events could be missed at other focal planes. To overcome these shortcomings, we present a modification of the classical microscope design with which two or more focal planes can be imaged simultaneously. In this design, the emission light collected by a single stationary objective lens is split into multiple channels. Light in each channel is focused on a CCD camera by a tube lens. By ensuring that the camera position with respect to the tube lens focal plane position is not the same in any two channels, distinct planes within the specimen can be simultaneously imaged. Here we discuss the implementation of a configuration with which four focal planes can be imaged simultaneously.© (2006) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.