DiscussionCells that form tissues interact through chemical and mechanical stimuli with adjacent cells and via the extracellular matrix. Highly interconnected communication network extend along all three dimensions and are lost in two-dimensional cultures. Fluorescence imaging of three-dimensional cell cultures imposes specific requirements: deep penetration into the specimen, high imaging speed and ultra-low intensity of the excitation light. Light sheet-based fluorescence microscopy offers a favorable solution for in vivo and in vitro tissue imaging. The talk illustrates the benefits of three-dimensional biology in the modern life sciences and suggests that LSFM is essential when investigating cellular and sub-cellular dynamic processes in three-dimensions over time and space.