Intracellular ion measurement with fluorescent indicators

Most of this book deals with electrical recording techniques which, although powerful, cannot always be used to study the composition of the intracellular environment particularly when small cells or populations of cells are the preparation of interest. Optical techniques are becoming an increasingly attractive alternative method because of their apparent non-invasive nature and ease of use. One area in which optical techniques have largely replaced other methods is the measurement of intracellular ion concentrations with fluorescent indicators. This chapter will provide an introduction to this increasingly important application and will also consider some of the basic features of optical versus electrical recording techniques. Electrical recording necessarily measures the movement of electrical charge. Individual charges can neither be created nor destroyed, and the overall balance between positive and negative charge must remain within narrow limits, since only a small charge separation causes a large electrical potential difference (voltage). Electrophysiology revolves around the measurement and/or control of charge separation and, as described elsewhere, the separation of charge by ion selective membranes allows determination of the ion levels within the cell as well as the creation of the Nernstian membrane potential. Some of the problems associated with electrical measurements arise from the fact that energy must be extracted from a system in order to ascertain its properties. Although careful electronic design can minimise the extraction of energy from the system (cell), the measurement process must perturb the system. In contrast to the idea of extracting energy from the system to measure its properties, with optical techiques (such as fluorescence and absorbance measurements) the experimenter actually supplies energy to examine the system, by supplying photons to the system and recording the results of the interaction of the photons with the system. This has several important consequences that should be kept in mind: (1) Photons will interact with any molecule that has absorbance in the