What are the advantages of using a ratiometric calcium probe vs. a single-channel dye?

The primary advantage of using ratiometric dyes as opposed to single-wavelength probes is that the ratio signal remains unaffected by variations in dye concentration, optical path length, and illumination intensity. This independence from these potential artifacts allows for the determination of intracellular calcium concentration with greater accuracy. Another key advantage is that ratiometric measures the ratio of fluorescence signals at two different wavelengths, while a single-channel dye only measures fluorescence at one wavelength. Additional advantages of ratiometric probes are that they reduce the effects of photobleaching, uneven dye loading, and leakage. Furthermore, ratiometric indicators help overcome difficulties associated with measuring calcium concentrations in cells that vary in thickness within a diverse population.