Fluorescence Digital Imaging Microscopy – Spatial Distribution of CA2+ and H+ in Single Cells

This chapter provides an introduction to fluorescent indicator dyes and digital imaging microscopy, and what information their combination has provided so far about the distribution of H⁺ and Ca²⁺ ions in living cells. A vital functional aspect that has not yet been addressed is that of the spatial resolution of information content of the recordings. For single-cell investigation, a method must be utilized that can "factor out" the dye concentration and path length requirements from the quantification relationship. Fluorescein, a fluorescent dye that has had many experimental uses, has a fluorescent spectrum that is altered with changes. The long history of fluorescent pH indicators illustrates both the difficulty in using them and the recognition of their potential utility, but useful fluorescent indicators were not available for other ions. Fluorescence digital imaging microscopy is the technique of digital imaging microscopy applied to fluorescent dyes, which provides both special opportunities and constraints in the digital imaging system.