FLIM-Based Intracellular and Extracellular pH Measurements Using Genetically Encoded pH Sensor
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.
Список научных проектов, где отмечена публикация
- Физиологическая роль гена insrr в регуляции работы панкреатических бета-клеток (7 Января 2019 года 31 Декабря 2021 года). . Грант, РФФИ.
- Создание и функциональный анализ генетически кодируемых сенсоров для определения внеклеточного рН in vitro и in vivo методом FLIM (6 Января 2020 года 31 Декабря 2022 года). . Грант, РФФИ.
- 19-73-20194 (6 Января 1970 года). . Внебюджетное финансирование.