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Updated: Jan 15, 2026

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Published on: December 7, 2015

Lysosomal pH Measurements via Dual-Imaging Microscopy.

Emily Kirkham¹, Sian Gardiner², Emyr Lloyd-Evans^1,2

¹School of Biosciences, Cardiff University, Cardiff, UK.

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

|October 13, 2025

Summary

This summary is machine-generated.

Dual-wavelength ratiometric fluorescent probes enable quantitative measurement of ion content in subcellular compartments. This chapter details using these probes for lysosomal pH measurement, covering selection, calibration, instrumentation, and high-throughput assay miniaturization.

Keywords:

Dual-wavelength ratio imaging Fluorescein isothiocyanate Fluorescent microscopy High-throughput LysoSensor Lysosome pH

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Published on: August 19, 2021

Area of Science:

Cell Biology
Biochemistry
Analytical Chemistry

Background:

Dual-wavelength ratiometric fluorescent probes are essential for quantifying ion content within subcellular compartments.
Lysosomal pH is a critical parameter influencing cellular processes and disease states.

Purpose of the Study:

To describe the application of fluorescent probes for accurate lysosomal pH measurement.
To provide a comprehensive guide for researchers utilizing these techniques.

Main Methods:

Selection of appropriate dual-wavelength ratiometric fluorescent probes.
Detailed calibration procedures for accurate pH determination.
Instrumentation setup for fluorescence measurements.
Miniaturization strategies for high-throughput screening.

Main Results:

Demonstration of quantitative lysosomal pH measurement using ratiometric probes.
Establishment of protocols for probe selection, calibration, and instrumentation.
Successful miniaturization of assays for high-throughput applications.

Conclusions:

Dual-wavelength ratiometric fluorescent probes offer a gold standard for quantitative subcellular ion measurements.
The described methods facilitate robust and high-throughput lysosomal pH determination.
This chapter serves as a valuable resource for researchers in cell biology and analytical chemistry.