Red/near‐infrared dyes are highly demanded for biological applications but most of them are far from satisfactory. In this work, a series of red/near‐infrared fluorophores based on electron‐withdrawing benzo[1,2‐b:4,5‐b′]dithiophene 1,1,5,5‐tetraoxide (BDTO) are synthesized and characterized. They possess both aggregation‐induced emission, and hybridized local and charge‐transfer characteristics. Crystallographic, spectroscopic, electrochemical and computational results reveal that the oxidation of benzo[1,2‐b:4,5‐b′]dithiophene to BDTO can endow the fluorophores with greatly red‐shifted emission, enhanced emission efficiency, reduced energy levels, enlarged two‐photon absorption cross section, and increased reactive oxygen species generation efficiency. The nanoparticles fabricated with a near‐infrared fluorophore TPA‐BDTO show high photostability and biocompatibility with good performance in targeted photodynamic ablation of cancer cells and two‐photon fluorescence imaging of intravital mouse brain vasculature.
Intravital fluorescence imaging of vasculature morphology and dynamics in the brain and in tumors with large penetration depth and high signal‐to‐background ratio (SBR) is highly desirable for the study and theranostics of vascular‐related diseases and cancers. Herein, a highly bright fluorophore (BTPETQ) with long‐wavelength absorption and aggregation‐induced near‐infrared (NIR) emission (maximum at ≈700 nm) is designed for intravital two‐photon fluorescence (2PF) imaging of a mouse brain and tumor vasculatures under NIR‐II light (1200 nm) excitation. BTPETQ dots fabricated via nanoprecipitation show uniform size of around 42 nm and a high quantum yield of 19 ± 1% in aqueous media. The 2PF imaging of the mouse brain vasculatures labeled by BTPETQ dots reveals a 3D blood vessel network with an ultradeep depth of 924 µm. In addition, BTPETQ dots show enhanced 2PF in tumor vasculatures due to their unique leaky structures, which facilitates the differentiation of normal blood vessels from tumor vessels with high SBR in deep tumor tissues. Moreover, the extravasation and accumulation of BTPETQ dots in deep tumor (more than 900 µm) is visualized under NIR‐II excitation. This study highlights the importance of developing NIR‐II light excitable efficient NIR fluorophores for in vivo deep tissue and high contrast tumor imaging.
Methods for noninvasive brain imaging are highly desirable to study brain structures in neuroscience. Two‐photon fluorescence microscopy (2PFM) with near‐infrared (NIR) light excitation is a relatively noninvasive approach commonly used to study brain with high spatial resolution and large imaging depth. However, most of the current studies require cranial window implantation or skull‐thinning methods due to attenuation of excitation light. 2PFM through intact mouse skull is challenging due to strong scattering induced by skull bone. Herein, NIR‐II light excitable single‐chain conjugated polymer dots (CPdots) with bright fluorescence in NIR‐I region (peak at ≈725 nm and quantum yield of 20.6 ± 1.0%) are developed for deep in vivo two‐photon fluorescence (2PF) imaging of intact mouse brain. The synthesized CPdots exhibit good biocompatibility, high photostability, and large two‐photon absorption cross section. The CPdots allow 2PF images acquired upon excitation at 800, 1040 and 1200 nm with the highest signal‐to‐background ratio of 208 demonstrated for 1200 nm excitation. Moreover, a 3D reconstruction of the brain blood vessel network is obtained with a large vertical depth of 400 µm through intact skull. This work demonstrates great potential of bright NIR fluorophores for in vivo deep tissue imaging.
Near infrared (NIR) light excitable photosensitizers are highly desirable for photodynamic therapy with deep penetration. Herein, a NIR‐II light (1200 nm) activated photosensitizer TQ‐BTPE is designed with aggregation‐induced singlet oxygen (1O2) generation for two‐photon photodynamic cancer cell ablation. TQ‐BTPE shows good two‐photon absorption and bright aggregation‐induced NIR‐I emission upon NIR‐II laser excitation. The 1O2 produced by TQ‐BTPE in an aqueous medium is much more efficient than that of commercial photosensitizer Ce6 under white light irradiation. Upon NIR‐II excitation, the two‐photon photosensitization of TQ‐BTPE is sevenfold higher than that of Ce6. The TQ‐BTPE molecules internalized by HeLa cells are mostly located in lysosomes as small aggregate dots with homogeneous distribution inside the cells, which favors efficient photodynamic cell ablation. The two‐photon photosensitization of TQ‐BTPE upon NIR‐I and NIR‐II excitation shows higher 1O2 generation efficiency than under NIR‐I excitation owing to the larger two‐photon absorption cross section at 920 nm. However, NIR‐II light exhibits better biological tissue penetration capability after passing through a fresh pork tissue, which facilitates stronger two‐photon photosensitization and better cancer cell ablation performance. This work highlights the promise of NIR‐II light excitable photosensitizers for deep‐tissue photodynamic therapy.
Two-photon fluorescence imaging allows in vivo study of biological structures and activities in deep tissues, in which bright fluorophores with high photostability and good biocompatibility are highly desirable. Herein, a small-molecule fluorogen with aggregation-induced emission (AIEgen) is complexed with fetal bovine serum (FBS) proteins to develop a protein-sized AIEgen-protein hybrid nanocomposite (TPEPy-FBS) with bright far-red/ near-infrared (NIR) emission, excellent photostability, and low phototoxicity for deep and high-resolution in vivo two-photon brain vasculature imaging. Upon complexation with FBS, the fluorescence of TPEPy is greatly intensified and a sixfold enhancement is observed with 10% FBS in aqueous media. The yielded TPEPy-FBS shows good physical stability in aqueous media and the phototoxicity of TPEPy is dramatically inhibited after complexation with FBS. Moreover, TPEPy-FBS exhibits bright two-photon fluorescence in far-red/ NIR region and good photostability upon femtosecond laser excitation, which facilitates high performance in vivo imaging. A large imaging depth of 656 µm is obtained in brain vasculature network imaging with a high signalto-background ratio of 234, where a small blood capillary of 1.05 µm can be resolved at an imaging depth of 656 µm. Highlighted is a simple and versatile strategy to develop efficient two-photon probes for in vivo biological imaging.
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