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Updated: Apr 17, 2026

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Published on: December 7, 2015

Measuring lysosomal pH by fluorescence microscopy.

Johnathan Canton¹, Sergio Grinstein²

¹Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario, M5G 0A4.

Methods in Cell Biology

|February 11, 2015

Summary

This summary is machine-generated.

Dual-wavelength ratio fluorescence microscopy offers a robust method for measuring organelle pH. This technique provides accurate, dynamic pH readings in subcellular compartments, overcoming limitations of single-wavelength approaches.

Keywords:

Dual-wavelength ratio imaging Lysosome fluorescein isothiocyanate fluorescence microscopy pH

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Area of Science:

Cell Biology
Biophysics
Microscopy

Background:

Accurate measurement of organelle pH is crucial for understanding cellular processes.
Traditional single-wavelength fluorescence methods are susceptible to artifacts like focal plane changes and dye bleaching.
A need exists for quantitative and dynamic pH measurement techniques in subcellular compartments.

Purpose of the Study:

To describe the application of dual-wavelength ratio fluorescence microscopy for measuring lysosomal pH.
To highlight the advantages and limitations of this advanced microscopy technique.
To provide detailed guidance on probe selection, calibration, and hardware for accurate pH measurements.

Main Methods:

Utilizing dual-wavelength ratio fluorescence microscopy for pH determination.
Implementing specific probe selection strategies tailored for organelle pH.
Performing detailed calibration methods to ensure quantitative accuracy.
Discussing essential hardware requirements for optimal performance.

Main Results:

Dual-wavelength ratio fluorescence microscopy provides quantitative and dynamic pH readouts.
This method is robust against variations in focal plane, dye volume, and fluorophore bleaching.
The technique is effectively applied to measure lysosomal pH.

Conclusions:

Dual-wavelength ratio fluorescence microscopy is a powerful and reliable tool for organellar pH measurement.
It offers significant advantages over single-wavelength methods, enabling precise subcellular pH analysis.
Detailed understanding of probe selection, calibration, and hardware is key to successful implementation.