A General Design of Compact “Zero‐On” NIR‐II Photoacoustic Dyes for High‐Fidelity Imaging

Abstract

ABSTRACT 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.