Merged molecular switches excel as optoacoustic dyes: azobenzene-cyanines are loud and photostable NIR imaging agents

Abstract

Optoacoustic imaging, also known as photoacoustic imaging, promises micron-resolution noninvasive imaging in biology at much deeper penetration (>cm) depths than e.g. fluorescence. However, the loud, photostable, NIR-absorbing molecular contrast agents which would be needed for optoacoustic imaging of enzyme activity remain unknown: most organic molecular contrast agents are simply repurposed fluorophores, with severe shortcomings of photoinstability or phototoxicity under optoacoustic imaging conditions, which are consequences of their slow S1→S0 electronic relaxation rates. We now disclose that known fluorophores can be rationally modified to reach ultrafast S1→S0 rates, without much extra molecular complexity, simply by merging them with molecular switches. Here, we merge azobenzene switches to cyanine dyes to give ultrafast relaxation (<10 ps, >100-fold faster). Even without adapting instrument settings, these azohemicyanine optoacoustic imaging agents deliver outstanding improvements in signal longevity (>1000-fold increase of photostability) and signal loudness (here: >3-fold even at time zero). We show why this still-unexplored design strategy can offer even stronger performance in the future, as a simple method that will also increase the spatial resolution and the quantitative linearity of photoacoustic response even over extended longitudinal imaging. By bringing the world of molecular switches and rotors to bear on unsolved problems that have faced optoacoustic agents, this practical strategy may be a crucial step towards unleashing the full potential, in fundamental studies and in translational uses, of optoacoustic imaging.