Ratiometric imaging of pH probes

Area of Science:

• Cell Biology
• Biochemistry
• Microscopy

Background:

• Intracellular pH (pHi) is a critical regulator of cellular processes.
• Accurate pHi measurement is essential for understanding cell function and disease.
• Existing methods for pHi determination have limitations in scope and resolution.

Purpose of the Study:

• To provide a comprehensive overview of ratiometric pH-sensitive probes for intracellular pH measurement.
• To discuss technical considerations for probe selection, application, and data analysis.
• To enable researchers to effectively measure pHi across various biological scales.

Main Methods:

• Discussion of technical aspects of ratiometric pH-sensitive dyes.
• Exploration of genetically encoded pH sensors.
• Protocols for probe loading into cells and tissues.
• Methods for ratiometric image acquisition and analysis.

Main Results:

• Ratiometric probes offer versatile tools for intracellular pH measurement.
• Probes are applicable across scales, from whole tissues to subcellular compartments.
• Standardized protocols facilitate reliable data generation.

Conclusions:

• Intracellular pH measurement is achievable with established ratiometric probe technologies.
• The described methods support diverse research applications in cell biology and beyond.
• This chapter serves as a practical guide for researchers utilizing pH-sensitive probes.