Highly specific C-C bond cleavage induced FRET fluorescence for in vivo biological nitric oxide imaging

A novel Förster resonance energy transfer (FRET) fluorescence "off-on" system based on the highly specific, sensitive and effective C-C bond cleavage of certain dihydropyridine derivatives was reported for real-time quantitative imaging of nitric oxide (NO). 1,4-Dihydropyridine was synthesized as a novel linker which could connect customized fluorophores and their corresponding quenchers. The specific and quantitative response to NO is confirmed using fluorescence spectrometry with the classical example of fluorescein isothiocyanate (FITC) and [4'-( , '-dimethylamino)phenylazo] benzoyl (DABCYL). The fluorescence intensity increased linearly with the increase in the amount of NO. Cells incubated with an exogenous NO donor emitted fluorescence as expected. A high fluorescence intensity was detected in macrophages which generate NO when incubated with lipopolysaccharide (LPS). The imaging shows about an 8-fold contrast between Freund's adjuvant stimulated feet and normal feet in mice after intravenous injection, which was the first example of semiquantitative fluorescence imaging of NO in mammals.