Chemigenetic Far-Red\nLabels and Ca<sup>2+</sup> Indicators\nOptimized for Photoacoustic Imaging

Photoacoustic imaging is an emerging modality with significant\npromise for biomedical applications such as neuroimaging, owing to\nits capability to capture large fields of view deep inside complex\nscattering tissue. However, widespread adoption of this technique\nhas been hindered by a lack of suitable molecular reporters for this\nmodality. In this work, we introduce chemigenetic labels and calcium\nsensors specifically tailored for photoacoustic imaging, using a combination\nof synthetic dyes and HaloTag-based self-labeling proteins. We rationally\ndesign and engineer far-red “acoustogenic” dyes, showing\nhigh photoacoustic turn-ons upon binding to HaloTag, and develop a\nsuite of tunable calcium indicators based on these scaffolds. These\nfirst-generation photoacoustic reporters show excellent performance\nin tissue-mimicking phantoms, with the best variants outperforming\nexisting sensors in terms of signal intensity, sensitivity, and photostability.\nWe demonstrate the application of these ligands for labeling HaloTag-expressing\nneurons in mouse brain tissue, producing strong, specifically targeted\nphotoacoustic signal, and provide a first example of <i>in vivo</i> labeling with these chemigenetic photoacoustic probes. Together,\nthis work establishes a new approach for the design of photoacoustic\nreporters, paving the way toward deep tissue functional imaging.