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Updated: Oct 21, 2025
Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy
Published on: August 19, 2021
Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric
Liam Wilkinson1, Johnathan Canton2
1Department of Biochemistry & Microbiology, Faculty of Science, University of Victoria; Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Science, University of Calgary.
Abstract:
In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.
Insights
New tools enable studying macropinosome maturation. Researchers can now assess pH, reactive oxygen species, and degradation in individual macropinosomes using ratiometric microscopy.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Macropinocytosis is crucial for immune cell homeostasis and immunity.
- Dysregulated macropinocytosis contributes to diseases like cancer and viral infections.
- Existing tools for studying macropinosome maturation are limited.
Purpose of the Study:
- To introduce novel tools for investigating the redox environment within macropinosomes.
- To detail methodologies for assessing macropinosome lumenal properties in live cells.
- To highlight the advantages of single-organelle measurements over population-based approaches.
Main Methods:
- Development of new tools for macropinosome research.
- Application of dual fluorophore ratiometric fluorescence microscopy.
- Measurement of pH, reactive oxygen species (ROS) production, and degradative capacity within individual macropinosomes.
Main Results:
- Demonstration of methodologies for live-cell analysis of macropinosome lumenal parameters.
- Characterization of spatiotemporal heterogeneity in macropinosome maturation.
- Validation of ratiometric microscopy for single organelle studies.
Conclusions:
- The developed tools provide unprecedented insights into macropinosome maturation dynamics.
- Ratiometric microscopy enables detailed analysis of individual macropinosomes, revealing heterogeneity.
- These advancements facilitate a deeper understanding of macropinocytosis in health and disease.
