Measurement of intracellular pH: differences between microspectrofluorimetric and imaging systems

Background. Since 1979, more than a thousand papers have reported the use of biscarboxyethylcarboxyfluorescein (BCECF) for making measurement of intracellular pH (pHi) using microspectrofluorimetric systems (MSFS). Looking critically at these papers, some odd and unexplained things can be observed. For example, there are big differences in the resting pHi of the cells, even in similar cells, in similar conditions and described in the same paper. Despite the huge development of imaging systems (IS), only few imaging measurements can be found in the literature to monitor real-time changes of pHi. Our aim was to compare MSFS (LIFE SCIENCES) and IS (OLYMPUS CellR).

Methods. pHi was measured in isolated pancreatic duct cells using the fluorescent dye BCECF. The nigericin/high K+ method was utilized to set the cells pHi to the same level and the fluorescence ratios (F490/F440 from which the pHi is calculated) were compared in MSFS and IS.

Results. Using IS, we found that there were groups of cells which were already saturated with the dye while others were not causing inhomogeneity between the fluorecent intensities of regions of interests (ROIs). These differences in the saturations lead to non-desirable deviations in ratios. Using MSFS, the inhomogeneity is not possible to detect, therefore, this can result in inaccurate calculation of pHi. However, the usage of IS allow to assign ROIs with almost identical homogeneities of fluorescence intensities. With using an IS one is capable of assigning 4–6 homogeneous ROIs at the same time. In conclusion, an imaging set-up should be prefered to measure pHi and more importantly, this change in practise would lead significantly less number of animals sacrified for scientific experiments. Supported by OTKA, MTA, OM.