Excitation-Matchable Shortwave Infrared Quinolinium Fluorophores: Decoding Spatiotemporal Interactions with Multiplexed Bioimaging.

Shortwave infrared (SWIR, 1000-2000 nm) imaging has emerged as an ideal window for multiplexed imaging at the mammalian level. However, this technology remains largely limited by the lack of a highly tunable "molecular scaffold core", which allows for the SWIR dye library to simultaneously meet high brightness, minimal cross-talk, and laser-compatible absorption. Herein, we introduce a diversified quinolinium-based "scaffold core" for the generation of the SWIR dye library, allowing excitation-matchable multiplexed imaging for decoding spatiotemporal interactions. This quinolinium domain enables establishing a series of SWIR heptamethine cyanines spanning maximum absorption wavelengths from 975 to 1046 nm. Among them, QC7-NEt2 and QC7-CN exhibit high brightness and orthogonal excitation at 980 or 1064 nm, matching well the accessible lasers. Using the orthogonal dye pair, we are able to conduct two- and even three-channel excitation-matchable multiplexed imaging with minimal cross-talk for decoding spatiotemporal interactions. These dyes demonstrated dynamic, high-resolution visualization of vasculature, lymph, and intestinal systems, especially for studying the interaction between deep-tissue organs and the surrounding vasculature networks. This study provides a full demonstration of our strategy in molecular design and streamlined SWIR dye discovery to push the limits of biological imaging in basic life science and clinical applications.