Szmacinski H, Gryczynski I, Lakowicz J R
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201, USA.
Biophys J. 1996 Jan;70(1):547-55. doi: 10.1016/S0006-3495(96)79601-7.
We report the calcium-dependent emission spectral properties of the calcium probe Indo-1 for three-photon excitation. We found that Indo-1 could be readily excited with the femtosecond pulses from a mode-locked Ti:sapphire laser at 885 nm. This wavelength is too long for two-photon excitation, which is expected to occur for wavelengths no longer than twice the longest single-photon absorption wavelength of 400 nm. For excitation at 885 nm the emission intensity was found to depend on the cube of the laser power, as expected for simultaneous interaction with three photons. At wavelengths below 840 nm the emission intensity depends on the square of the laser power, indicating two-photon excitation at shorter wavelengths. The intensity decays of Indo-1 were found to be dependent on Ca2+ and essentially identical for one- and three-photon excitation. The emission anisotropy of Indo-1 was found to be considerably higher for three-photon excitation than for one-photon excitation, consistent with cos6 theta photoselection, as compared with cos2 theta photoselection for one-photon excitation. The high values of the anisotropy are in agreement with those expected for a three-photon process. Calcium-dependent emission spectra were observed for Indo-1 with three-photon excitation, demonstrating that three-photon excitation of Indo-1 can be used for calcium imaging by emission intensity ratio measurements. The calcium-dependent emission spectra indicate a higher three-photon cross-section for the calcium-free form of Indo-1 than for the calcium-bound form. The possible advantages of three-photon excitation include the availability of the appropriate wavelengths with solid-state lasers, enhanced spatial resolution due to a reduced size of the excited volume, absence of light quenching, and possibly high selectivity of the three-photon excitation process.
我们报告了钙探针吲哚-1在三光子激发下的钙依赖性发射光谱特性。我们发现，锁模钛宝石激光器产生的飞秒脉冲在885nm波长下可轻松激发吲哚-1。该波长对于双光子激发来说太长了，双光子激发预计发生在波长不超过最长单光子吸收波长400nm两倍的情况下。在885nm激发时，发现发射强度取决于激光功率的立方，这与同时与三个光子相互作用的预期一致。在波长低于840nm时，发射强度取决于激光功率的平方，表明在较短波长下为双光子激发。发现吲哚-1的强度衰减取决于Ca2+，并且单光子激发和三光子激发的衰减基本相同。发现吲哚-1的发射各向异性在三光子激发时比单光子激发时高得多，与单光子激发的cos2θ光选择相比，这与cos6θ光选择一致。各向异性的高值与三光子过程的预期值一致。在三光子激发下观察到了吲哚-1的钙依赖性发射光谱，表明吲哚-1的三光子激发可用于通过发射强度比测量进行钙成像。钙依赖性发射光谱表明，吲哚-1的无钙形式比钙结合形式具有更高的三光子截面。三光子激发的可能优点包括固态激光器可提供合适的波长、由于激发体积减小而提高的空间分辨率、不存在光猝灭以及三光子激发过程可能具有的高选择性。
