Much effort has been devoted to the generation of fluorescent probes by synthetic approaches. In this study, we developed a facile strategy to construct far-red fluorescent probes based on through-space charge transfer within complexes of acceptors and donors and their "twist + twist" interactions. Owing to their rare two-photon excitation property, the complexes could be used for in vivo imaging of the mouse cerebrovascular system.

High spatial resolution, low background, and deep tissue penetration have made near-infrared II (NIR-II) fluorescence imaging one of the most critical tools for in vivo observation and measurement. However, the relatively short retention time and potential toxicity of synthetic NIR-II fluorophores limit their long-term application. Here, we report the use of infrared fluorescent proteins (iRFPs) as in vitro and in vivo NIR-II probes permitting prolonged continuous imaging (up to 15 months). As a representative example, iRFP713 is knocked into the mouse genome to generate a transgenic model to allow temporal and/or spatial expression control of the probe. To demonstrate its feasibility in a genuine diagnostic context, we adopt two liver regeneration models and successfully track the process for a week. The performance and monitoring efficacy are comparable to those of μCT and superior to those of indocyanine green dye. We are also able to effectively observe the pancreas, despite its deep location, under both physiological and pathological conditions. These results indicate that the iRFP-assisted NIR-II fluorescence system is suitable for monitoring various tissues and in vivo biological processes, providing a powerful noninvasive long-term imaging platform.

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Simple donor−acceptor (D−A) molecules with large Stokes-shifted and bright near-infrared (NIR) emissions are extremely attractive for the high-resolution three-photon fluorescence (3PF) imaging of deep tissues. Herein, we present a new strategy for significantly extending the Stokes shifts of D−A fluorophores, relying on increasing the excited-state structural/electronic relaxation of the donor moiety. The prototype of the D−A structure DPCN, containing the planar donor N,N′diphenyl-dihydrophenazine (DHP) and the acceptor malononitrile, exhibits normal Stokes-shifted (∼60 nm) NIR emission. By introducing two methyl groups at the ortho sites of the fused DHP ring of DPCN, DMPCN (methyl-decorated DPCN) is developed with an obviously bent donor unit. Compared to DPCN, DMPCN exhibits significantly blueshifted absorption and keeps NIR luminescence with a large Stokes shift of ∼200 nm, mainly due to the remarkable bent-to-planar transformation of the donor upon photoexcitation. Additionally, the DMPCN NPs offer enhanced luminescence quantum yields (11%) and three-photon excitation cross sections. More importantly, DMPCN NPs exhibit excellent performances for in vivo 3PF imaging of the cerebrovascular tissue and lipid droplets (LDs) in a fatty liver with a depth of 1200 and 50 μm, respectively. This research provides a distinct strategy for extending the Stokes shifts of D−A fluorophores, inspiring the utilization of dynamic NIR fluorophores for in vivo 3PF imaging.