Intracellular potassium is a critical element to many vital cell processes. Thus, the ability to accurately measure intracellular concentration of K+ is essential. Because the behavior of intracellular fluorescent probes is different from those in a buffer, they have to be calibrated in situ. The standard approach to calibrate intracellular ion probes is based on the use of ionophores with the idea to equalize the intra- and extracellular concentrations. However, such equalization can only be achieved when the membrane potential y is zero. Complete cell depolarization requires cell treatment with metabolic poisons; because of their toxicity, such treatments are not universally used, in which case the results may be affected by residual y.
Therefore, we set out to develop a different method based on fluorescence widefield and confocal microscopy. Instead of trying to depolarize cells, we measure their actual y and use this information to calculate intracellular [K+]. That enables us to construct complete calibration curves (the relationship between fluorescent signal and intracellular K+).
At this point we have worked out the main experimental steps. We apply mitochondrial uncoupler CCCP to obtain the redistribution of the potentiometric dye TMRE between the cytosol and the environment to findy, valinomycin to increase K+ permeability in the presence of buffers with varying [K+] and ratiometric K-sensitive probe PBFI.
Successful development of this method will open new possibilities in cell research. We intend to use it to clarify the role of K+ in apoptosis. The method can be also extended to other intracellular ions, such as calcium.
The ability to accurately measure intracellular concentration of K+ is essential for many cell biological applications. Because the behavior of intracellular fluorescent probes is different from those in a buffer, they have to be calibrated in situ. The standard calibration method is based on the use of ionophores to equalize intracellular and extracellular ion concentrations. However, artifacts are possible when using this approach.
Therefore, we set out to develop a different method utilizing fluorescence widefield and confocal microscopy. We add a fluorescent potentiometric dye to determine the cell membrane potential and use this information to calculate intracellular [K+]. That enables us to achieve more accurate calibration and measurements of intracellular potassium. This approach can be extended to other intracellular ions.