title: Measuring lysosomal pH by fluorescence microscopy.
abstract: 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.
authors: Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario, M5G 0A4.
authors: Johnathan
authors: J
authors: Canton
authors: Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, M5C 1N8, Canada.
authors: Sergio
authors: S
authors: Grinstein
mesh_terms: D000818:Animals; D002138:Calibration; D002460:Cell Line; D003911:Dextrans / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D013194:Staining and Labeling
chemical_list: D003911:Dextrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; C052526:fluorescein-dextran
keywords: Dual-wavelength ratio imaging; Lysosome; fluorescein isothiocyanate; fluorescence microscopy; pH