Measuring Phagosomal pH by Fluorescence Microscopy.
Dual wavelength ratiometric imaging has become a powerful tool for the study of pH in intracellular compartments. It allows for the dynamic imaging of live cells while accounting for changes in the focal plane, differential loading of the fluorescent probe, and photobleaching caused by repeated image acquisitions. Ratiometric microscopic imaging has the added advantage over whole population methods of being able to resolve individual cells and even individual organelles. In this chapter we provide a detailed discussion of the basic principles of ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, the necessary instrumentation, and calibration methods.
Program in Cell Biology, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, 686 Bay Street, 19-9800, Toronto, ON, Canada, M5G 0A4.
Johnathan
J
Canton
Program in Cell Biology, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, 686 Bay Street, 19-9800, Toronto, ON, Canada, M5G 0A4. sergio.grinstein@sickkids.ca.|Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, 290 Victoria Street, Toronto, ON, Canada, M5C 1N8. sergio.grinstein@sickkids.ca.
Sergio
S
Grinstein
D000818:Animals; D015374:Biosensing Techniques; D002138:Calibration; D006801:Humans; D006863:Hydrogen-Ion Concentration; D021621:Imaging, Three-Dimensional; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D009895:Opsonin Proteins / Q000378:metabolism; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism*; D000067996:RAW 264.7 Cells; D013194:Staining and Labeling; D015054:Zymosan / Q000378:metabolism
D009895:Opsonin Proteins; D015054:Zymosan
Dual wavelength ratio imaging; Fluorescein; Fluorescence microscopy; Oregon Green; Phagosome; SNARF1; pH