High-resolution visualization of the deep brain is still a challenging and very significant issue. Multiphoton microscopy (MPM) holds great promise for high-spatiotemporal deep-tissue imaging under NIR-III and NIR-IV excitation. However, thus far, their applications have been seriously restricted by the scarcity of efficient organic probes. Herein, we designed and synthesized two donor-acceptor-donor-type conjugated small molecules (TNT and TNS) for in vivo mouse deep-brain imaging with three- and four-photon microscopy under 1700 and 2200 nm excitation. With a selenium (Se) substitution, we synthesized two conjugated small molecules to promote their emission into the deep near-infrared region with high quantum yields of 55% and 20% in THF solvent, respectively, and their water-dispersive nanoparticles have relatively large absorption cross-sections in the 1700 and 2200 nm windows, respectively, with good biosafety. With these superiorities, these organic NPs achieve high-resolution deep-brain imaging via three-photon and four-photon microscopy with excitation at 1700 and 2200 nm windows, and 1620 μm deep in the brain vasculature can be visualized in vivo. This study demonstrates the efficiency of NIR-emissive conjugated small molecules for high-performance MPM imaging in the NIR-III and NIR-IV window and provides a route for the future design of organic MPM probes.
Keywords: 1700 nm window; 2200 nm window; NIR-II; conjugated oligomer; four-photon imaging; multiphoton microscopy.