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Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy
Published on: April 28, 2014
Measuring Phagosomal pH by Fluorescence Microscopy
Gerone A Gonzales1, Johnathan Canton2,3
1Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.
Abstract:
Dual-wavelength and dual-fluorophore ratiometric imaging has become a powerful tool for the study of pH in intracellular compartments. It allows for the dynamic imaging of live cells while accounting for changes in the focal plane, differential loading of the fluorescent probe, and photobleaching caused by repeated image acquisitions. Ratiometric microscopic imaging has the added advantage over whole-population methods of being able to resolve individual cells and even individual organelles. In this chapter, we provide a detailed discussion of the basic principles of ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, the necessary instrumentation, and calibration methods.
Insights
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.
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.
