Generalization of the Ratiometric Method to Extend pH Range Measurements of the BCECF Probe

There is a variety of fluorescent probes for pH measurements and which are mainly used for biological systems. In general, they can be classified into two groups. The first group includes fluorescent pH probes which exhibit a single fluorescence emission peak. For these probes, the fluorescence excitation profile is pH-dependent and the shape of the emission spectra remains almost constant. Hence, the ratiometric pH measurement-which makes pH determination independent of the probe concentration-is implemented when the excitation is performed at two excitation wavelengths and the fluorescence emission is measured at one wavelength. The second group exhibits a dual fluorescence emission peak. Here, each protonated or deprotonated form exhibits characteristics emission and/or absorption spectra. Shifts between spectra obtained for protonated and deprotonated species can be exploited in order to perform a ratiometric measurement. In this study we present a methodology that evaluates the precision of the ratiometric measurements based on multiple wavelengths excitation to determine the optimum wavelengths combination for pH determination in biological samples. This methodology using the BCECF probe is applied to measure the pH drift in cell culture medium. It exhibits a high precision and significantly extends the range of validity for pH measurements spanning from very acidic to basic.