Photolysis of caged calcium in femtoliter volumes using two-photon excitation

A new technique for the determination of the two-photon uncaging action cro ss section (delta(u)) of photolyzable calcium cages is described. This tech nique is potentially applicable to other caged species that can be chelated by a fluorescent indicator dye, as well as caged fluorescent compounds. Th e two-photon action cross sections of three calcium cages, DM-nitrophen, NP -EGTA, and azid-1, are studied in the range of excitation wavelengths betwe en 700 and 800 nm, Azid-1 has a maximum delta(u) of similar to 1.4 GM at 70 0 nm, DM-nitrophen has a maximum delta(u) of similar to 0.013 GM at 730 nm, and NP-EGTA has no measurable uncaging yield. The equations necessary to p redict the amount of cage photolyzed and the temporal behavior of the liber ated calcium distribution under a variety of conditions are derived. These equations predict that by using 700-nm light from a Ti:sapphire laser focus ed with a 1.3-NA objective, essentially all of the azid-1 within the two-ph oton focal volume would be photolyzed with a 10-mu s pulse train of similar to 7 mW average power. The initially localized distributions of free calci um will dissipate rapidly because of diffusion of free calcium and uptake b y buffers. in buffer-free cytoplasm, the elevation of the calcium concentra tion at the center of the focal volume is expected to last for similar to 1 65 mu s.