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Anal Bioanal Chem (2013) 405:39833987 DOI 10.1007/s00216-013-6860-y
RESEARCH PAPER
The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging
Sandrine Poa-Guyon & Hlne Pasquier & Fabienne Mrola &
Nicolas Morel & Marie Erard
Received: 6 January 2013 /Revised: 20 February 2013 /Accepted: 21 February 2013 /Published online: 10 March 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.
Keywords Enhanced cyan fluorescent protein or ECFP . pH sensor . Fluorescent protein . FLIM . PC12 cells . Granular pH
Introduction
The pH of intracellular compartments is finely regulated and modulates many cell functions [13]. Numerous tools have been developed to measure intracellular pHs and understand the interconnections between H+ concentrations and physio-pathological processes [1, 2]. Among them, sensitive fluorescence techniques are nondestructive to living cells and easy to implement on an optical microscope. Nevertheless, the quantitative analysis of pH (or more generally of any physicochemical parameter) in a cell with a classical wide field fluorescence microscope remains hindered by the difficulty to quantify fluorescence intensities for each individual. The latter are indeed proportional (a) to the chromophore concentration inside the cell which is difficult to control and (b) to the cell thickness which determines the length of the excitation...