Photolysis of Caged Calcium in Femtoliter Volumes Using Two-Photon Excitation

@article{citeulike:3037890, abstract = {A new technique for the determination of the two-photon uncaging action cross section ([delta]u) of photolyzable calcium cages is described. This technique is potentially applicable to other caged species that can be chelated by a fluorescent indicator dye, as well as caged fluorescent compounds. The two-photon action cross sections of three calcium cages, DM-nitrophen, NP-EGTA, and azid-1, are studied in the range of excitation wavelengths between 700 and 800 nm. Azid-1 has a maximum [delta]u of \~{}1.4 GM at 700 nm, DM-nitrophen has a maximum [delta]u of \~{}0.013 GM at 730 nm, and NP-EGTA has no measurable uncaging yield. The equations necessary to predict the amount of cage photolyzed and the temporal behavior of the liberated calcium distribution under a variety of conditions are derived. These equations predict that by using 700-nm light from a Ti:sapphire laser focused with a 1.3-NA objective, essentially all of the azid-1 within the two-photon focal volume would be photolyzed with a 10-{micro}s pulse train of \~{}7 mW average power. The initially localized distributions of free calcium will dissipate rapidly because of diffusion of free calcium and uptake by buffers. In buffer-free cytoplasm, the elevation of the calcium concentration at the center of the focal volume is expected to last for \~{}165 {micro}s.}, author = {Brown, Edward B. and Shear, Jason B. and Adams, Stephen R. and Tsien, Roger Y. and Webb, Watt W. }, citeulike-article-id = {3037890}, journal = {Biophys. J.}, keywords = {2pm, caged, calcium, photolysis, photon, technique, two}, month = {January}, number = {1}, pages = {489--499}, posted-at = {2008-07-23 23:31:58}, priority = {2}, title = {Photolysis of Caged Calcium in Femtoliter Volumes Using Two-Photon Excitation}, url = {http://www.biophysj.org/cgi/content/abstract/76/1/489}, volume = {76}, year = {1999} }

TY - JOUR ID - citeulike:3037890 L3 - citeulike-article-id:3037890 TI - Photolysis of Caged Calcium in Femtoliter Volumes Using Two-Photon Excitation JF - Biophys. J. VL - 76 IS - 1 SP - 489 EP - 499 N2 - A new technique for the determination of the two-photon uncaging action cross section ([delta]u) of photolyzable calcium cages is described. This technique is potentially applicable to other caged species that can be chelated by a fluorescent indicator dye, as well as caged fluorescent compounds. The two-photon action cross sections of three calcium cages, DM-nitrophen, NP-EGTA, and azid-1, are studied in the range of excitation wavelengths between 700 and 800 nm. Azid-1 has a maximum [delta]u of ~1.4 GM at 700 nm, DM-nitrophen has a maximum [delta]u of ~0.013 GM at 730 nm, and NP-EGTA has no measurable uncaging yield. The equations necessary to predict the amount of cage photolyzed and the temporal behavior of the liberated calcium distribution under a variety of conditions are derived. These equations predict that by using 700-nm light from a Ti:sapphire laser focused with a 1.3-NA objective, essentially all of the azid-1 within the two-photon focal volume would be photolyzed with a 10-{micro}s pulse train of ~7 mW average power. The initially localized distributions of free calcium will dissipate rapidly because of diffusion of free calcium and uptake by buffers. In buffer-free cytoplasm, the elevation of the calcium concentration at the center of the focal volume is expected to last for ~165 {micro}s. KW - 2pm KW - caged KW - calcium KW - photolysis KW - photon KW - technique KW - two AU - Brown, Edward AU - Shear, Jason AU - Adams, Stephen AU - Tsien, Roger AU - Webb, Watt PY - 1999/01/01/ UR - http://www.biophysj.org/cgi/content/abstract/76/1/489 ER -