ABSTRACT
Ion indicators consist of fluorophores coupled to ion binding functional groups known as chelators in such a way that ion binding to the chelator is transduced into a proportional change in the fluorescence of the fluorophore. Cell loading and response calibration are the most prominent technical challenges involved in calcium imaging with fluorescent indicators. Fluorescent protein-based pH indicators exploit the fact that green fluorescent protein itself is pH-sensitive, due to ionization of the phenol group of its 4-(p-hydroxybenzylidene)imidazolidin-5-one fluorophore. The difference in the extracellular and intracellular solvent environments of the probe results in a change of its fluorescence quantum yield, producing a fluorescence change that is coupled to the membrane potential. Indeed, the indirect nature of the response mechanism creates a liability for generating false-positive responses. Differences between the expression and activity levels of enzymes are often physiologically consequential, leading to the need to measure both quantities in parallel.
