It is of great significance to study the brain structure and function in deep-tissue for neuroscience research and bio-medical applications because of the urgent demand for precise theranostics. Three-photon fluorescence microscopic (3PFM) bioimaging excited by the light in near-infrared IIb (NIR-IIb, 1,500-1,700 nm) spectral region is one of the most promising imaging techniques with the advantages of high spatial resolution, large imaging depth, and reduced scattering. Herein, a type of NIR-IIb light excitable deep-red emissive semiconducting polymer dots (P-dots) with bright 3PF and large three-photon absorption cross-section (σ₃) at 1,550 nm was prepared. Then the P-dots were functionalized with polystyrene polymer polystyrene graft ethylene oxide functionalized with carboxyl groups (PS-PEG-COOH) and modified with NH₂-poly(ethylene glycol) (PEG) to synthesis photochemically stable and biocompatible P-dots nanoparticles (NPs). Further the P-dots NPs were utilized for in vivo 3PFM bioimaging of cerebral vasculature with and without the brain skull under 1,550 nm femtosecond (fs) laser excitation. In vivo 3PFM bioimaging of the mice cerebral vasculature at various vertical depths was obtained. Moreover, a vivid three-dimensional structure of the mice vascular architecture beneath the skull was reconstructed. At the depth of 350 μm beneath the brain skull, 3.8 µm blood vessels could still be clearly recognized. NIR-IIb excitable P-dots assisted 3PFM bioimaging has great potential in accurate deep tissue bioimaging.

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.

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.