Photolysis of caged calcium in femtoliter volumes using two-photon excitation
Photolysis of caged calcium in femtoliter volumes using two-photon excitation
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DOI：
10.1016/s0006-3495(99)77217-6
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发表时间：
1999-01-01
影响因子：
3.4
通讯作者：
Webb, WW
中科院分区：
文献类型：
--
作者：
Brown, EB;Shear, JB;Webb, WW
本文介绍了一种测定光解钙笼的双光子释放作用截面的新技术。这种技术是潜在的适用于其他笼物种，可以螯合的荧光指示剂染料，以及笼荧光化合物。在700 ~ 800 nm激发波长范围内，研究了三种钙笼（DM-nitrophen，NP-EGTA和azid-1）的双光子作用截面，Azid-1在700 nm处的最大Δ（u）约为1.4 GM，DM-nitrophen在730 nm处的最大Δ（u）约为0.013 GM，NP-EGTA没有可测量的解笼产率。所需的方程来预测笼光解的量和时间的行为，释放的钙分布在各种条件下的推导。这些方程预测，通过使用来自用1.3-NA物镜聚焦的Ti：蓝宝石激光器的700-nm光，双光子聚焦体积内的基本上所有叠氮化物-1将用类似于7 mW平均功率的10 μ s脉冲序列光解。由于游离钙的扩散和缓冲液的吸收，游离钙的初始局部分布将迅速消失。在无缓冲液的细胞质中，在焦点体积中心处钙浓度的升高预计持续类似于165 μ s。
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 similar to 1.4 GM at 700 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 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-mu s pulse train of similar to 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 similar to 165 mu s.