Dongyu Li

Toxicity assessment and long-term three-photon fluorescence imaging of bright aggregation-induced emission nanodots in zebrafish

Aggregation-induced emission (AIE) luminogen displays bright fluorescence and has photobleaching resistance in its aggregation state. It is an ideal fluorescent contrast agent for bioimaging. Multiphoton microscopy is an important tool for bioimaging since it possesses the ability to penetrate deep into biological tissues. Herein, we used AIE luminogen together with multiphoton microscopy for long-term imaging of zebrafish. A typical AIE luminogen, 2,3-bis(4-(phenyl(4- (1,2,2-triphenylvinyl) phenyl)amino)phenyl) fumaronitrile (TPE-TPA-FN or TTF), was encapsulated with 1,2-distearoyl-sn-glycero-3-phosphoethanola-mine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000) to form nanodots that exhibited bright three-photon fluorescence under 1,560 nm-femtosecond (fs) laser excitation. The TTF-nanodots were chemically stable in a wide range of pH values and showed no in vivo toxicity in zebrafish according to a series of biological tests. The TTF-nanodots were microinjected into zebrafish embryos, and the different growth stages of the labeled embryos were monitored with a three-photon fluorescence microscope. TTF-nanodots could be traced inside the zebrafish body for as long as 120 hours. In addition, the TTF-nanodots were utilized to target the blood vessel of zebrafish, and three-photon fluorescence angiogram was performed. More importantly, these nanodots were highly resistant to photobleaching under 1,560 nm-fs excitation, allowing long-term imaging of zebrafish.

Aggregation-induced emission nanoparticles as photosensitizer for two-photon photodynamic therapy

Two-photon excited fluorescence microscopy and nanoparticle-assisted photodynamic therapy (PDT) are two important areas in biomedical research, and their combination can be more beneficial. A type of red emissive photosensitizer (PS) with aggregation-induced emission (AIE) features, which is called tetraphenylethylene (TPE-red), was synthesized and further encapsulated with poly(styrene-co-maleic anhydride) (PSMA) to form nanoparticles. Two-photon fluorescence, as well as two-photon excited reactive oxygen species (ROS) generation by TPE-red–PSMA nanoparticles, was characterized. A large two-photon absorption cross-section was observed at 1040 nm with a femtosecond (fs) laser. PDT via two-photon excitation was well realized on tumor cells, using TPE-red–PSMA nanoparticles as PSs under 1040 nm fs laser excitation. Based on our study, we believe that two-photon excited PDT with AIE-active PSs has great potential applications in deep tissue imaging-guided therapy.

Synthesis, two-photon absorption and aggregation-induced emission properties of multi-branched triphenylamine derivatives based on diketopyrrolopyrrole for bioimaging

In this work, three new diketopyrrolopyrrole (DPP)-based multi-branched derivatives (YJ-1, YJ-2 and YJ-3) with triphenylamine, 2,4,6-tri([1,1′-biphenyl]-4-yl)-1,3,5-triazine and 2,2′,2′′-(nitrilotr-is([1,1′-biphenyl]-4′,4-diyl))tris(3-phenylacrylonitrile) cores have been designed and synthesized. Their one- and two-photon absorption properties have been investigated. The two-photon absorption cross sections (σ) measured by the open aperture Z-scan technique are determined to be 2912, 2016 and 2800 GM for YJ-(1–3), respectively. This result indicates that donor–acceptor–donor (D–A–D)-type molecules are benefit to improve σ and their σ data increase with the better intramolecular charge transfer (ICT). Also, all of the three DPP derivatives exhibit good aggregation-induced emission (AIE) properties which are very weakly fluorescent in DMF, but a strong red fluorescent emission in solid state and in the aggregate state. More importantly, diketopyrrolopyrrole with tri-phenylamine (YJ-1) was applied for cell imaging and two-photon excited fluorescence in vivo imaging of mouse ear.

Aggregation-Induced Emission Luminogen with Near-Infrared-II Excitation and Near-Infrared-I Emission for Ultradeep Intravital Two-Photon Microscopy

Currently, a serious problem obstructing the large-scale clinical applications of fluorescence technique is the shallow penetration depth. Two-photon fluorescence microscopic imaging with excitation in the longer-wavelength near-infrared (NIR) region (>1100 nm) and emission in the NIR-I region (650-950 nm) is a good choice to realize deep-tissue and high-resolution imaging. Here, we report ultradeep two-photon fluorescence bioimaging with 1300 nm NIR-II excitation and NIR-I emission (peak ∼810 nm) based on a NIR aggregation-induced emission luminogen (AIEgen). The crab-shaped AIEgen possesses a planar core structure and several twisting phenyl/naphthyl rotators, affording both high fluorescence quantum yield and efficient two-photon activity. The organic AIE dots show high stability, good biocompatibility, and a large two-photon absorption cross section of 1.22 × 103 GM. Under 1300 nm NIR-II excitation, in vivo two-photon fluorescence microscopic imaging helps to reconstruct the 3D vasculature with a high spatial resolution of sub-3.5 μm beyond the white matter (>840 μm) and even to the hippocampus (>960 μm) and visualize small vessels of ∼5 μm as deep as 1065 μm in mouse brain, which is among the largest penetration depths and best spatial resolution of in vivo two-photon imaging. Rational comparison with the AIE dots manifests that two-photon imaging outperforms the one-photon mode for high-resolution deep imaging. This work will inspire more sight and insight into the development of efficient NIR fluorophores for deep-tissue biomedical imaging.

Aggregation-induced emission luminogen-assisted stimulated emission depletion nanoscopy for super-resolution mitochondrial visualization in live cells

Aggregation-induced emission luminogens (AIEgens) are fluorescent agents that are ideal for bioimaging and have been widely used for organelle targeting, cellular mapping, and tracing. Owing to their promising characteristics, AIEgen-based nanoparticles have recently been used for the stimulated emission depletion (STED) super-resolution imaging of fixed cells. In the present study, and for the first time, we used an AIEgen for dynamic STED nanoscopic imaging of a specific organelle in live cancer cells. TPA-T-CyP is a synthetic red&NIR-emitting luminogen with AIE features that can spontaneously and specifically aggregate on mitochondria without the need for encapsulation or surface modification. The STED efficiency of aggregated TPA-T-CyP can reach more than 80%, and super-resolution imaging of TPA-T-CyP-stained mitochondria in live HeLa cells is possible, with a lateral spatial resolution of 74 nm. We found that TPA-T-CyP enabled the dynamic visualization of mitochondria, and the motion, fusion, and fission of mitochondria were clearly observable on a super-resolution scale. AIEgen-based super-resolution organelle visualization has great potential for many basic biomedical studies.

Short-wave infrared emitted/excited fluorescence from carbon dots and preliminary applications in bioimaging

Fluorescent carbon dots (FCDs) have attracted tremendous attention in biological applications. The short-wave infrared (SWIR, 900–1700 nm) spectral range is considered as a novel optical tissue window due to low photon scattering. In this work, we investigated the fluorescence characteristics of FCDs based on the SWIR spectral range. SWIR emissions were observed from FCDs for the first time, when long-wavelength excitation (e.g. longer than 731 nm) was adopted. Wavelength-tunable two-photon fluorescence could also be obtained from the FCDs, under 1000–1560 nm SWIR femtosecond (fs) excitation. Interestingly, when an fs excitation wavelength as long as 1560 nm was adopted, two typical nonlinear optical signals, namely two-photon fluorescence and third harmonic generation (THG), could be observed. Based on the one-photon SWIR fluorescence, FCDs were successfully utilized for in vivo sentinel node mapping and tumour imaging. Under SWIR fs excitation, FCD-assisted two-photon fluorescence microscopy realized deep-tissue imaging of zebrafish embryos and the brain neuron networks of mice. SWIR excited and emitting FCDs have potential as fluorescent probes for deep-tissue and high-contrast functional bioimaging and related applications in the future.