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Updated: Jul 31, 2026
09:46
Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy
Published on: April 28, 2014
15.6K
Lysosomal pH Measurements via Dual-Imaging Microscopy
Emily Kirkham1, Sian Gardiner2, Emyr Lloyd-Evans1,2
1School of Biosciences, Cardiff University, Cardiff, UK.
Methods in Molecular Biology (Clifton, N.J.)
|October 13, 2025
Summary
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.
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.
Related Concept Videos
03:22Strong Acid and Base Solutions
A strong acid is a compound that dissociates completely in an aqueous solution and produces a concentration of hydronium ions equal to the initial concentration of acid. For example, 0.20 M hydrobromic acid will dissociate completely in water and produces 0.20 M of hydronium ions and 0.20 M of bromide ions.
03:27Mixtures of Acids
The pH of a solution containing an acid can be determined using its acid dissociation constant and its initial concentration. If a solution contains two different acids, then its pH can be determined using one of several methods depending upon the relative strength of the acids and their dissociation constants.
A Mixture of a Strong Acid and a Weak Acid
In a mixture of a strong acid and a weak acid, the strong acid dissociates completely and becomes a source of almost all the hydronium ions...
A Mixture of a Strong Acid and a Weak Acid
In a mixture of a strong acid and a weak acid, the strong acid dissociates completely and becomes a source of almost all the hydronium ions...
02:28Titration Calculations: Strong Acid - Strong Base
Calculating pH for Titration Solutions: Strong Acid/Strong Base
A titration is carried out for 25.00 mL of 0.100 M HCl (strong acid) with 0.100 M of a strong base NaOH. The pH at different volumes of added base solution can be calculated as follows:
(a) Titrant volume = 0 mL. The solution pH is due to the acid ionization of HCl. Because this is a strong acid, the ionization is complete and the hydronium ion molarity is 0.100 M. The pH of the solution is then:
A titration is carried out for 25.00 mL of 0.100 M HCl (strong acid) with 0.100 M of a strong base NaOH. The pH at different volumes of added base solution can be calculated as follows:
(a) Titrant volume = 0 mL. The solution pH is due to the acid ionization of HCl. Because this is a strong acid, the ionization is complete and the hydronium ion molarity is 0.100 M. The pH of the solution is then:
01:19Mixtures of Acids
The pH of a solution containing an acid can be determined using its acid dissociation constant and initial concentration. If a solution contains two different acids, then its pH can be determined using one of several methods depending on the relative strength of the acids and their dissociation constants.
In a strong and weak acid mixture, the strong acid dissociates completely and becomes a source of almost all the hydronium ions present in the solution. In contrast, the weak acid shows...
In a strong and weak acid mixture, the strong acid dissociates completely and becomes a source of almost all the hydronium ions present in the solution. In contrast, the weak acid shows...