Photoacoustic lifetime contrast between methylene blue monomers and self-quenched dimers as a model for dual-labeled activatable probes

Abstract. Activatable photoacoustic probes efficiently combine the high spatial resolution and penetration depth of ultrasound with the high optical contrast and versatility of molecular imaging agents. Our approach is based on photoacoustic probing of the excited-state lifetime of methylene blue (MB), a fluorophore widely used in clinical therapeutic and diagnostic applications. Upon aggregation, static quenching between the bound molecules dramatically shortens their lifetime by three orders of magnitude. We present preliminary results demonstrating the ability of photoacoustic imaging to probe the lifetime contrast between monomers and dimers with high sensitivity in cylindrical phantoms. Gradual dimerization enhancement, driven by the addition of increasing concentrations of sodium sulfate to a MB solution, showed that lifetime-based photoacoustic probing decreases linearly with monomer concentration. Similarly, the addition of 4 mM sodium dodecyl sulfate, a concentration that amplifies MB aggregation and reduces the monomer concentration by more than 20-fold, led to a signal decrease of more than 20 dB compared to a solution free of surfactant. These results suggest that photoacoustic imaging can be used to selectively detect the presence of monomers. We conclude by discussing the implementation of the monomer–dimer contrast mechanism for the development of an enzyme-specific activatable probe.