The carboxylic acid of 7-hydroxy-4-methylcoumarin-3-acetic acid can be activated in an aqueous solution with a water-soluble carbodiimide such as EDAC (E2247) in order to couple this fluorophore to amines, hydrazines or hydroxylamines on biomolecules of interest to create blue-fluorescent bioconjugates that can be used as a contrasting color in multicolor applications with excitation/emission maxima ~360/455 nm. Because the fluorescence may not be as bright as that of other dyes or may be obscured by autofluorescence, hydroxycoumarin derivatives are only recommended for use with highly abundant targets. Unlike AMCA and Alexa Fluor® 350 dye hydroxycoumarin exhibits pH-sensitive spectra.