PMID: 11908850Mar 23, 2002Paper

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

European Biophysics Journal : EBJ
Nikolai I KiskinDavid Ogden

Abstract

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 microm radius vesicles, measured with one-photon excitation during two-photon photolysis by repetitive 10 micros laser exposu...Continue Reading

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