Photolysis of caged calcium in femtoliter volumes using two-photon excitation.
A new technique for the determination of the two-photon uncaging action cross section (deltau) 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 deltau of approximately 1.4 GM at 700 nm, DM-nitrophen has a maximum deltau of approximately 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-micros pulse train of approximately 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 approximately 165 micros.
Brown
E B
EB
Developmental Resource for Biophysical Imaging and Optoelectronics, Cornell University, Ithaca, New York 14853, USA.
Shear
J B
JB
Adams
S R
SR
Tsien
R Y
RY
Webb
W W
WW
D000085:Acetates / Q000528:radiation effects*; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000528:radiation effects*; D002614:Chelating Agents / Q000528:radiation effects*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000528:radiation effects; D005029:Ethylenediamines / Q000528:radiation effects*; D007700:Kinetics; D008956:Models, Chemical; D010782:Photolysis; D017785:Photons
D000085:Acetates; D002614:Chelating Agents; D005029:Ethylenediamines; C057014:DM-nitrophen; C085112:2-nitrophenyl-EGTA; D004533:Egtazic Acid; D002118:Calcium