The efficiency of two-photon photolysis of a "caged" fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals

Localized photolysis of caged neurotransmitters with the two-photon effect for investigations at synaptic preparations was evaluated by determining the toxicity to synaptic transmission of pulsed near-IR laser light focused into the terminals of the snake neuromuscular junction, and measuring the extent of photolysis of a conventional caging group with similar irradiation in microcuvette experiments. Photodamage was seen in synaptic terminals as a large, irreversible increase of spontaneous synaptic activity with laser flashes of 5 ms at 1 Hz at average powers > 5 mW and was due to multiphoton absorption. Localized photolysis due to two-photon absorption was investigated for a representative caged fluorophore, the 1-(2-nitrophenyl)ethyl ether of pyranine (NPE-HPTS). Irradiation of NPE-HPTS at 5 mW with the same optical arrangement produced very low rates of photolysis. NPE-HPTS photolysis mechanisms were investigated at high laser powers by measuring (1) the kinetics of two-photon fluorescence generated by two-photon photolysis in the focal volume and (2) the rates of HPTS accumulation inside closed 2-10 μm radius vesicles, measured with one-photon excitation during two-photon photolysis by repetitive 10 μs laser exposures. The two-photon crosssection of NPE-HPTS photolysis calculated from the rates is 0.02-0.04 GM (10^-50 cm⁴×s/photon) and limits the efficiency of photolysis at 5 mW. With free diffusional exchange, 50% steady-state cage depletion in the focal volume was estimated to occur only at high laser powers of ca. 72 mW, masked in experiments by multiphoton bleaching. Based on these results, the two-photon photolysis cross-section needed for 50% steady-state photolysis of a caged neurotransmitter at 5 mW is calculated as 31 GM, much higher than in existing caged compounds.