Application of two-photon flash photolysis to measure microscopic diffusion and calcium fluxes

摘要

Two-photon excitation (TPE) via a microscope objective lens poduces a spatially confined excitation volume where UV-excited caged molecules may be broken (uncaged) to release active products. We describe an optical system that creates a stationary parfocal TPE uncaging spot on the stage of a conventional confocal microscope. With this system, we have examined the ability of two dyes to track microscopic calcium changes produced by TPE photolysis of DM-nitrophen. We find that, even when EGTA is used with a low affinity indicator, the dye signals are complicated by diffusion of both indicator-Ca complex and CaEGTA to produce a signal that does not simply report the spatial dimensions of the calcium release site. In addition, the time course of calcium release is poorly reported. This suggests that considerable caution must be applied to the interpretation of spatially resolved calcium signals inside cells. We have also used TPE of CMND-caged fluorescein to measure the rate of fluorescein production in test solution (2500 s~1) as well as the diffusion of fluorescein in drops of solution and within and between between eye lens fiber cells. While diffusion of uncaged fluorescein was about an order of magnitude slower inside fiber cells than in aequeous solution, slower diffusion between cells could also be detected and could be explained by the gap junctions joining the cells behaving as a barrier to diffusion. By using a computer model, parameter fits to experimental data gave estimates for both intracellular and intercellular diffusion coefficients. From this anaysis, the gap junctions in eye lens fiber cells permit exchange of low molecular weight compounds between cells at about 0.4% of the rate of free diffusion.