Fig. 1: Strategies for ratiometric probes with characteristic analysis.

a FRET ratiometric probes: excitation/emission cross-talk. b Unimolecular ratiometric probes: different photobleaching rates and concentration-dependent diffusion between reactant and product fluorophores in dynamic cellular environment. c In this work, anti-Kasha-active ratiometric probes enabling accurate and reliable ratiometric quantification: an invariant NIR emission from the Kasha-based S1 state serve as an internal reference; a green emission from the anti-Kasha-active S2 state linearly increase with the concentration of targeted analyte, due to the rapid radiative process from S2 to S0 state, competing with the internal conversion (IC) from S2 to S1 state.