Structurally Modulated Formation of Cyanine J‐aggregates with Sharp and Tunable Spectra for Multiplexed Optoacoustic and Fluorescence Bioimaging

J‐aggregation brings intriguing optical and electronic properties to molecular dyes and significantly expands their applicability across diverse domains, yet the challenge for rationally designing J‐aggregating dyes persists. Herein, we developed a large number of J‐aggregating dyes from scratch by progressively refining structure of a common heptamethine cyanine. J‐aggregates with sharp spectral bands (full‐width at half‐maximum ≤ 38 nm) are attained by introducing a branched structure featuring a benzyl and a trifluoroacetyl group at meso‐position of dyes. Fine‐tuning the benzyl group enables spectral regulation of J‐aggregates. Analysis of single crystal data of nine dyes reveals a correlation between J‐aggregation propensity and molecular arrangement within crystals. Some J‐aggregates are successfully implemented in multiplexed optoacoustic and fluorescence imaging in animals. Notably, three‐color multispectral optoacoustic tomography imaging with high spatiotemporal resolution is achieved, owing to the sharp and distinct absorption bands of the J‐aggregates.