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Updated: Jul 25, 2025

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Published on: December 7, 2015

Measuring Phagosomal pH by Fluorescence Microscopy.

Gerone A Gonzales¹, Johnathan Canton^2,3

¹Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.

Methods in Molecular Biology (Clifton, N.J.)

|June 26, 2023

Summary

This summary is machine-generated.

Dual-wavelength, dual-fluorophore ratiometric imaging enables dynamic, live-cell pH studies in organelles. This advanced microscopy technique overcomes limitations of other methods for precise intracellular pH measurements.

Keywords:

CypHer5e Dual-fluorophore ratio imaging Dual-wavelength ratio imaging Fluorescein Fluorescence microscopy Oregon Green Phagosome SNARF1 pH pHrodo

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Last Updated: Jul 25, 2025

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Published on: December 7, 2015

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

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Published on: April 5, 2017

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Published on: April 28, 2014

Area of Science:

Cell biology
Microscopy
Biophysics

Background:

Ratiometric imaging uses dual wavelengths and fluorophores for accurate pH measurements in live cells.
It corrects for variations in probe concentration and photobleaching, improving data reliability.
This technique offers superior resolution compared to whole-population methods, enabling single-cell and organelle analysis.

Purpose of the Study:

To detail the principles of dual-wavelength, dual-fluorophore ratiometric imaging.
To explain its application in measuring phagosomal pH.
To guide researchers on probe selection, instrumentation, and calibration.

Main Methods:

Utilizing dual-wavelength and dual-fluorophore microscopy.
Applying ratiometric analysis to correct for experimental variations.
Focusing on live-cell imaging of intracellular compartments, specifically phagosomes.

Main Results:

Demonstration of ratiometric imaging's capability for dynamic pH monitoring in live cells.
Accounting for focal plane shifts, probe loading, and photobleaching.
Successful application to phagosomal pH measurement.

Conclusions:

Dual-wavelength, dual-fluorophore ratiometric imaging is a robust method for intracellular pH studies.
The technique provides high resolution for individual cellular components.
Detailed guidance on implementation, including probe choice and calibration, is provided.