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Published on: June 7, 2019
The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging
Sandrine Poëa-Guyon1, Hélène Pasquier, Fabienne Mérola
1Université of Paris Sud, Orsay, France.
Analytical and Bioanalytical Chemistry
|March 12, 2013
Summary
This study highlights enhanced cyan fluorescent protein (ECFP) as a sensitive tool for measuring intracellular pH. Fluorescence lifetime imaging with ECFP enables precise pH monitoring in cellular compartments, advancing live-cell imaging techniques.
Area of Science:
- Cell Biology
- Biophysics
- Biochemistry
Background:
- Intracellular pH is critical for cellular functions, including protein stability and metabolism.
- Genetically encoded fluorescent proteins are used to monitor intracellular pH.
- Existing pH sensors often use green/yellow fluorescent proteins, limiting multicolor applications.
Purpose of the Study:
- To evaluate enhanced cyan fluorescent protein (ECFP) as a pH sensor using fluorescence lifetime imaging.
- To demonstrate the utility of ECFP for quantitative intracellular pH measurements.
- To analyze pH dynamics in specific cellular compartments.
Main Methods:
- Utilized fluorescence lifetime imaging microscopy (FLIM).
- Engineered ECFP fusions with chromogranin A for targeting secretory granules.
- Applied ammonium chloride to induce pH changes in living PC12 cells.
Main Results:
- ECFP exhibited significant pH sensitivity (32% lifetime change) between pH 5 and 7.
- Achieved pH measurement accuracy better than 0.2 pH units.
- Successfully monitored intragranular pH in PC12 cells and analyzed kinetic variations.
Conclusions:
- ECFP is a robust and sensitive genetically encoded pH sensor.
- FLIM-based ECFP sensors are suitable for precise, quantitative intracellular pH monitoring.
- This approach facilitates the study of pH dynamics in live cells and specific organelles.
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