A ratiometric imaging method for mapping ion flux densities.

A heterogeneous distribution of ion channels on the cell surface is a prerequisite for several cellular functions. Thus, there has been considerable interest in methods allowing the mapping of ion channel distributions. Here we report on a novel ratiometric imaging technique appropriate to measure spatially resolved ion flux signals by using ion sensitive dyes. However, given that certain relevant cell properties like the surface to volume ratio may exhibit significant spatial heterogeneities, the local influx signal cannot be interpreted as a measure of the local open channel concentration or flux density. To overcome this problem, we suggest an internal normalization procedure, which, in analogy to, but clearly distinct from, well-established ratioing techniques, eliminates effects which would otherwise obscure the desired result. Ratioing is performed on flux signals from a given cell, triggered by two different, subsequent stimuli. If the two stimuli address different ion channels, the flux density distribution caused by two channel types can be determined relative to each other. In cases where one of the stimuli triggers a spatially homogeneous flux signal, ratioing yields an ion flux density map for a given channel type. Thus distribution patterns of ion channels active during a given stimulus may be derived.