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Molecular imaging of biological analytes provides detailed insights into signaling processes. Ratiometric probes are particularly attractive due to the ability to quantify analyte production. However, design strategies for ratiometric probes can be hindered by spectral overlap of the product and reactant species. In this chapter, we provide protocols for the synthesis and application of RF620, a ratiometric probe for H2O2 displaying dramatic changes in both excitation and emission wavelengths, designed using an approach we term chemosele