FLIM-Based Intracellular and Extracellular pH Measurements Using Genetically Encoded pH Sensor

Area of Science:

• Cell Biology
• Physiology
• Biophysics

Background:

• Accurate pH determination in live cells and tissues is crucial for understanding physiological and cellular processes.
• Existing methods may have limitations in measuring extracellular pH in specific ranges.

Purpose of the Study:

• To create and validate SypHerExtra, a genetically encoded fluorescent sensor for extracellular pH measurement.
• To demonstrate the utility of SypHerExtra and SypHer3s for quantitative intracellular and extracellular pH monitoring.

Main Methods:

• Development of SypHerExtra by fusing the SypHer3s pH sensor with a neurexin transmembrane domain.
• Utilizing fluorescence lifetime imaging microscopy (FLIM) for pH-dependent fluorescence lifetime measurements.
• Live eukaryotic cell imaging to assess sensor performance.

Main Results:

• Both SypHer3s and SypHerExtra exhibited pH-dependent fluorescence lifetimes when excited at 445 nm.
• SypHerExtra successfully measured extracellular pH in live eukaryotic cells.
• SypHer3s was effective for intracellular pH measurements.

Conclusions:

• SypHerExtra is a viable tool for quantitative extracellular pH measurements in the mildly alkaline range (pH 7.5-9.5).
• SypHer3s is suitable for intracellular pH measurements within the same range.
• These genetically encoded sensors, combined with FLIM, offer a powerful method for dual intracellular and extracellular pH determination in biological systems.