Chapter forty-five - Photoactivation of Fluorescence as a Probe for Cytoskeletal Dynamics in Mitosis and Cell Motility

Abstract

Photoactivation of fluorescence represents a novel technology for marking structures in living cells and organisms with the precision of a light microbeam. This chapter discusses the use of this novel fluorescence marking technique for studying cytoskeletal polymer dynamics. In principle, photoactivation is similar to fluorescence redistribution after photobleaching (FRAP), but there is a difference. While photobleaching involves the generation of a non-fluorescent mark on a bright background, photoactivation generates a fluorescent mark on a dark background. This is achieved by “caging” a fluorochrome with a photolabile group that renders it non-fluorescent. After the incorporation of this tagged molecule into structures of interest, local activation of fluorescence is done by a brief exposure to an ultraviolet (UV) microbeam, which is followed by time-lapse fluorescence video microscopy. This chapter describes the structure and properties of caged fluorochromes synthesized in the laboratory as well as the design of a computer-controlled microscope to study cytoskeletal dynamics. Photoactivation of fluorescence is expected to have wide applicability as a non-perturbing marking technique. It will be used in studying membrane dynamics, embryonic fate mapping, and intracellular membrane transport, among other areas.