Molecular rotors are loud, highly photostable, NIR/SWIR-active molecular optoacoustic contrast agents

Optoacoustic or photoacoustic imaging (PA) combines optical excitation with acoustic readout, for non-invasive in vivo imaging at up to several centimetres' penetration depth, and down to micron resolution. Conceptually, many chromophore types can be used for simple anatomical PA, where signal generation is the only requirement: but few can perform the more complex task of molecular imaging of enzyme activity in practice, for which the many requirements include enzymatic signal switch-on. Here, we leverage molecular rotors to give a rational blueprint for high-performance small molecule PA contrast agents in the NIR/SWIR biotransparency window that offer straightforward adaptation for molecular imaging. According to our hypothesis, the ultrafast nonradiative S1→S0 kinetics (knr) of triphenylmethane rotors would be the chemical key to their PA signal (loudness) being strong, linear against imaging intensity, and outstandingly photostable. After we identified a route to shift typically the green/red absorbance of triarylmethanes into the NIR/SWIR, we showed that they are indeed >1000-fold more photostable as well as >5-fold louder than typical reference chromophores for PA. Pioneering femtosecond transient absorption spectroscopy results in live cells, as a bridge from spectroscopy to biology, supported our conceptual approach of maximising knr to optimise the several key practical aspects of PA performance. Much like molecular switches, molecular rotors had only been used in a limited scope of imaging modalities to date. This approach now shows the potential of rotors for quantitative longitudinal PA; and more broadly, the results will guide the future of mechanism-based design in rationally improving dye performance in a range of basic and translational imaging methods.