Novel approach to real-time flash photolysis and confocal [Ca²⁺] imaging

Flash photolysis of "caged" compounds using ultraviolet light is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. Studies that employ this technique have used diverse strategies for controlling the spatial and temporal application of light to the specimen. In this paper, we describe a new system for flash photolysis that delivers light from a pulsed, adjustable intensity laser through an optical fiber coupled into the epifluorescence port of a commercial confocal microscope. Photolysis is achieved with extremely brief (5 ns) pulses of ultraviolet light (355 nm) that can be synchronized with respect to confocal laser scanning. The system described also localizes the UV intensity spatially so that uncaging only occurs in defined subcellular regions; moreover, because the microscope optics are used in localization, the photolysis volume can be easily adjusted. Experiments performed on rat ventricular myocytes loaded with the Ca²⁺ indicator fluo-3 and the Ca²⁺ cage o-nitrophenyl ethylene glycol bis(2-aminoethyl ether)-N,N,N′N′-tetraacetic acid (NP-EGTA) demonstrate the system's capabilities. Localized intracellular increases in [Ca²⁺] can trigger sarcoplasmic reticular Ca ²⁺ release events such as Ca²⁺ sparks and, under certain conditions, regenerative Ca²⁺ waves. This relatively simple and inexpensive system is, therefore, a useful tool for examining local signaling in the heart and other tissues.