The ability of dyes—notably litmus, phenolphthalein and phenol red—to change their color in response to a pH change has found widespread application in research and industry. Fluorescent dyes, however, provide the increased sensitivity required for optical pH measurements inside live cells. They also offer much greater spatial sampling capability when compared with microelectrode techniques. These advantages have spurred the development of improved fluorescent dyes that can sense pH changes within physiological ranges. Of course, many of the same fluorescent pH indicators can also be used as pH sensors in cell-free media.
To quantitatively measure pH, it is essential to match the indicator's pKa to the pH of the experimental system. Consequently, the following two sections of this chapter are divided into pH indicators for use in environments with near-neutral pH (Probes Useful at Near-Neutral pH—Section 20.2) and pH indicators for use in relatively acidic environments (Probes Useful at Acidic pH—Section 20.3). Intracellular pH is generally between ~6.8 and 7.4 in the cytosol and ~4.5 and 6.0 in the cell's acidic organelles. Unlike intracellular free Ca2+ concentrations, which can rapidly change by perhaps 100-fold, the pH inside a cell varies by only fractions of a pH unit, and such changes may be quite slow.
We offer a variety of fluorescent pH indicators, pH indicator conjugates and other reagents for pH measurements in biological systems. Among these are several probes with unique optical response and specialized localization characteristics:
- Visible light–excitable SNARF pH indicators enable researchers to determine intracellular pH in the physiological range using dual-emission or dual-excitation ratiometric techniques (Probes Useful at Near-Neutral pH—Section 20.2), thus providing important tools for confocal laser-scanning microscopy and flow cytometry.
- pHrodo dye and LysoSensor probes, for use in acidic environments such as lysosomes (Probes Useful at Acidic pH—Section 20.3).
- Fluorescent pH indicators coupled to dextrans are extremely well retained in cells, do not bind to cellular proteins and have a reduced tendency to compartmentalize (pH Indicator Conjugates—Section 20.4).
Families of Molecular Probes pH indicators are listed in Molecular Probes pH indicator families, in order of decreasing pKa—Table 20.1 in approximate order of decreasing pKa value.