Chapter 16 - Ratio Imaging: Practical Considerations for Measuring Intracellular Ca2+ and pH in Living Cells

This chapter discusses the use of ratiometric fluorescent probes for measuring intracellular pH (pH(i)) and Ca-i(2+) concentration at the single cell level. The development of sensitive and stable probes for monitoring pH(i) and Ca-i(2+) in living cells has provided the scientists with invaluable tools for studying a multitude of cellular processes. These probes afford a noninvasive and semiquantitative assessment of pH(i) and Ca-i(2+), eliminating the need to impale cells with microelectrodes. The development and availability of membrane permeant Ca-i(2+)- and pH-specific fluorescent probes coupled to major advances in the technology and design of low-light-level charge-coupled devices geared toward biological applications, and improved microscope optics, have made it possible to visualize a two-dimensional fluorescence signal that is related to Ca-i(2+) and pH(i). The chapter describes the basis for using dual excitation ratio imaging and tries to provide a framework for understanding and developing the technique for investigating the roles of Ca-i(2+) and pH(i) in cellular processes. The technique of quantitative ratio imaging for the measurement of pH(i) and Ca-i(2+) has revolutionized the field of cell physiology. Using the proper equipment and choosing the right dyes for the experimental needs should provide reliable and reproducible results. More importantly, the amount of data produced from each experiment, when analyzing pH(i) and Ca-i(2+) on an individual cell basis, yields valuable information on the heterogeneity of cellular responses.