Microfluorometric Imaging of Time and Space Dependent Calcium Concentration Changes in Mammalian Neurons

Abstract

Excellent signal to noise ratio and resolution has made fluorescence microscopy a favorite tool for molecular probing in the biosciences. Cytoplasmic indicator molecules allow monitoring of intracellular ion concentration changes with high spatial and temporal resolution even in cells not dissociated from their surrounding tissue (tissue slice). General interest has focused especially on intracellular calcium, an ubiquitous second messenger whose many vital functions are well known. Detailed knowledge of the spatial intracellular calcium dynamics appears to be a key for the understanding of the parallel functioning and specificity of the many pathways utilizing Ca²⁺ as a second messenger. Consequently, development of high efficiency fluorescent indicators concentrated to some extent on Ca²⁺ as the target ion. Today, fluorescent indicators are available for a series of ions and molecules including H⁺ (pH), C1⁻ and Mg²⁺ (see Tsien, 1989) and more indicators become available almost monthly. The following treatment is limited to the measurement of calcium, although the basic considerations are generally the same for the measurement of other target ions.