20] Two-photon activation of caged calcium with submicron, submillisecond resolution

Abstract

Publisher Summary This chapter describes equipment for the two-photon excitation (TPE) of caged calcium and delineates the spatial resolution of calcium release possible with this apparatus. There are many possible uses for TPE of calcium cages. The competition of buffers for locally released calcium could provide a window into the spatial heterogeneities of the intrinsic calcium-buffering dynamics in living cells. This could be accomplished through utilization of the relative uptake of liberated calcium ions by a fluorescent calcium indicator and intrinsic calcium buffers as a measure of the intrinsic buffering rate and capacity of cells. The chapter shows that for at least one cell type, the kinetics should happen significantly faster than the diffusional escape of calcium ions from the TPE focal volume, allowing these buffering properties to be measured with submicron, three-dimensional resolution and thereby extending previous whole cell studies. These measurements and calculations demonstrate that complete two-photon release of calcium with high three-dimensional spatial resolution is possible and can be an extremely useful tool in the study of a variety of cellular processes.