Disclosed are imaging agents having the following Formula I: (I)； wherein F is a near infrared fluorophore, S is an enzymatically cleavable oligopeptide, Q is a fluorescence quencher molecule, and M is a moiety selected from the group consisting of PEG or derivative thereof and a targeting ligand, and wherein F, Q and M are linked to separate amino acids of the enzymatically cleavable oligopeptide. Compositions comprising such compounds, as well as methods of use, methods of identifying a cell or a population of cells in vivo expressing a protease of interest, and methods of treating a disease through imaging are also disclosed.
