Measurement of intracellular Ca²⁺ concentration using Indo-1 during simultaneous flash photolysis to release Ca²⁺ from DM-nitrophen

We have constructed a modular instrument to measure intracellular [Ca²⁺] ([Ca²⁺]_i) in single isolated cells while simultaneously imposing step changes in [Ca²⁺]_i using "caged Ca²⁺". By combining the outputs of a xenon arc lamp with a frequency-tripled (Nd: YAG) laser, the instrument can operate with low maintained illumination to measure [Ca²⁺]_i using a ratiometric Ca²⁺-sensitive fluorophore and also activate the release of Ca²⁺ from a caged-Ca²⁺ compound with a high energy pulse of ultraviolet light. This instrument is simple to assemble, introduces little electrical noise, provides a wide range of illumination power, produces only moderate photobleaching of the Ca²⁺ indicator and can be readily adapted to diverse cellular preparations. We demonstrate the use of this system to measure step changes in [Ca²⁺]_i in adult rat ventricular myocytes and a human embryonic kidney cell line (293 cells) in culture.