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Measuring Phagosomal pH by Fluorescence Microscopy

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Phagocytosis and Phagosomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1519))

Abstract

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.

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Acknowledgments

J.C. was supported by a Cystic Fibrosis Canada postdoctoral fellowship. Research in our laboratory is supported by grants FDN-143202 and MOP-126069 from the Canadian Institutes of Health Research.

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Authors and Affiliations

  1. 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 Canton & Sergio Grinstein

  2. Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, 290 Victoria Street, Toronto, ON, Canada, M5C 1N8

    Sergio Grinstein

Authors
  1. Johnathan Canton
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  2. Sergio Grinstein
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Correspondence to Sergio Grinstein .

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Editors and Affiliations

  1. Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada

    Roberto Botelho

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Canton, J., Grinstein, S. (2017). Measuring Phagosomal pH by Fluorescence Microscopy. In: Botelho, R. (eds) Phagocytosis and Phagosomes. Methods in Molecular Biology, vol 1519. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6581-6_12

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  • DOI: https://doi.org/10.1007/978-1-4939-6581-6_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6579-3

  • Online ISBN: 978-1-4939-6581-6

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