Introduction

Intracellular pH (pHi) has been studied extensively for over a century using a wide range of techniques. These techniques have been subject to constant improvements to the extent that useful measurements can now be made in even the smallest of cells. This chapter outlines the development of ion-sensitive microelectrodes and dyes and highlights some of the difficulties and dangers of both of these methods. It then concentrates on some recent advances in our understanding of how fluorescent pH-sensitive dyes might best be used. Finally, a tabulated comparison between the two techniques is presented.

Historical perspective on pHi measurement

Although pHi changes had been observed much earlier, the first measurements of what could loosely be described as intracellular pH were made around 1910 using cell extracts and platinum/hydrogen electrodes (for a review see Caldwell, 1956). For example, in 1912 Michaelis and Davidoff measured the pH of blood and noted that red cell lysis caused a change in bulk pH. The technique of cell lysis was perhaps most suited to the measurement of pHi in non-nucleated erythrocytes where intracellular compartments did not complicate the measurements. At around the same time various workers were using naturally occurring pH indicators to visualise changes in pHi (e.g. Crozier, 1918). The problems associated with such indirect techniques were recognised very early on and the search for better methods led in three directions: distribution of weak acids and bases; smaller electrodes to allow direct measurement of pHi; and better indicators and techniques to load them into cells.