Measuring lysosomal pH by fluorescence microscopy.

The technique of dual-wavelength ratio fluorescence microscopy provides a powerful tool to measure organellar pH. Unlike single-wavelength measurements, this method is unaffected by changes in focal plane, dye volume, and fluorophore bleaching, providing a quantitative and dynamic readout of the pH of subcellular compartments. This chapter describes the application of dual-wavelength ratio fluorescence microscopy to the measurement of lysosomal pH, highlighting its advantages and limitations. Probe selection, calibration methods, and salient aspects of the required hardware are discussed in detail.