A General Design of Compact "Zero-On" NIR-II Photoacoustic Dyes for High-Fidelity Imaging.

Near-infrared-II (1000-1700 nm) photoacoustic imaging enables deep-tissue visualization with high spatial precision but lacks organic probes integrating general design, reduced molecular complexity, and high-fidelity in vivo signal output. Herein, we present a general building-block design for ultralow-molecular-weight "zero-on" near-infrared-II photoacoustic dyes for in vivo high-fidelity imaging. Constructed around a dimethyl-dihydroacridine scaffold and synthesized in three steps, these dyes incorporate biomarker-responsive blocks for targeted activation and absorption-expanded blocks for near-infrared-II absorption tuning, while maintaining a molecular weight <500 Da. This general building-block design enables tailored applications. Ultralow molecular weight improves solubility, tissue penetration and clearance. Notably, biomarker activation drives nonconjugated-to-conjugated structural transition, inducing >700 nm absorption redshift and boosting near-infrared-II photoacoustic signals at 1064 nm from levels statistically indistinguishable from water (p > 0.05, defining "zero" probe background) to an 89.8-fold enhancement in "on" state. "Zero" background minimizes false positives from misinterpreting probe background accumulation signals in diseased tissues as biomarker activation, even in high-uptake organs like the liver. In a blinded study, our "zero-on" probes identified early‑stage hepatotoxicity in mice with 100% accuracy, outperforming 85% accuracy of traditional non-zero background "off-on" probes. This work enables a general design to achieve ultralow-molecular-weight near-infrared-II photoacoustic dyes for high-fidelity signal output in vivo.