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

Biosensors (Basel). 2021 Sep 15;11(9):340. doi: 10.3390/bios11090340.


The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems.

Keywords: FLIM; GFP; SypHer3s; SypHerExtra; extracellular pH; fluorescent proteins; intracellular pH; pH; pH sensor.

MeSH terms

  • Biosensing Techniques*
  • Fluorescence
  • Green Fluorescent Proteins
  • Humans
  • Hydrogen-Ion Concentration
  • Microscopy, Fluorescence


  • Green Fluorescent Proteins