Simplifying ratiometric C-SNARF-1 pH calibration procedures with a simple post-processing

Abstract

International audienceA simple and easy to implement numerical method is proposed in order to considerably simplify the experimental calibration procedure of C-SNARF-1 indicator used for ratiometric pH sensing. Usually, calibration is based on the measurement of fluorescence spectra using perfectly calibrated equipment at extreme pH values. The calibration solutions must be extremely well controlled in terms of indicator concentration and path length. Also, the optical equipment used must be well controlled and excitation energy as well as fluorescence collection efficiency must be perfectly constant over the whole calibration procedure. The method we propose is based on the fact that the emission fluorescence energy does not only depend on pH but also on the excitation wavelength. In this paper, we propose a model describing the evolution of the emitted energy as a function of pH and excitation wavelength. We show that the emitted energy evolves linearly with pH and we express this linear evolution as a function of the excitation wavelength. We also show the evolution of the isosbestic (or isoemitting) point as a function of the excitation wavelength. Knowing the linear dependence of the emitted energy as a function of excitation wavelength allows post-processing calibration spectra obtained with basic optical equipment where the excitation energy, fluorescence collection efficiency, indicator concentration and path length can vary over the calibration session. Because the calibration procedure becomes independent of the above mentioned parameters, the post-processing we propose considerably simplify indicators calibration. This method can easily be transposed, not only to other ratiometric pH indicators, but also to ion sensing fluorescent indicators exhibiting dual emission peaks