3PF AIE first point noninvasive imaging microscopy, to achieve visualization of the process in vivo stroke

Vascular system is a key component of the circulatory system, for in vivo visualization of cerebral vascular research by fluorescent nanoprobes important for understanding common brain diseases. Two-photon fluorescence (2PF) of the microscope due to its traditional near infrared excitation and a low tendency to photobleaching, commonly used for deep tissue imaging in vivo. However, even by means of a cranial window, since the excitation light scattering in the biological matrix beam, usually limited to the imaging 2PF shallow depth imaging. In addition, due to the permanent loss of the whole skull, resulting in low survival rate after surgery animals. Brain tissue inflammation and disturbance of the natural environment is unavoidable destructive interference, resulting in reduced image quality. The natural environment of the brain is the ideal way to study brain diseases Therefore, the observation of non-invasive methods. To solve this problem, three-photon fluorescence (3PF) microscopy imaging as an effective imaging modality is growing rapidly. Different from the conventional imaging 2PF, 3PF imaging techniques using near-infrared region (1000-1700 nm) of higher order nonlinear localized excitation, increased penetration depth significantly, spatial and temporal resolution and letter background ratio (SBR). Despite these advantages, a major limitation is the lack 3PF image having a large three-photon absorption cross section and a high fluorescence quantum efficiency organic dyes. Recently, Tang Benzhong Fellow Hong Kong University of Science and Technology, Sun Yat-Sen Liang Guodong, Associate Professor and Professor Qian Jun Zhejiang University to cooperate on \” Advanced Materials \” published an article entitled \”Facile Synthesis of Efficient Luminogens with AIE Features for Three-Photon Fluorescence Imaging of the Brain through the Intact Skull \”article reported their progress in 3PF organic dye imaging area. They synthesized solid light emitting material emitting a significant induction of aggregation (the AIE) properties and a high quantum yield (42.6%) by a simple reaction scheme. Far red / near-infrared BTF synthetic molecule having a bright emission points AIE can be prepared by simple precipitation process nm. AIE point having a high brightness, a large Stokes shift, good biocompatibility, good light stability and a large three-photon absorption cross section, can be used as an effective fluorescent nanoparticleProbes for in vivo imaging of cerebrovascular complete skull through the three-photon fluorescence imaging microscopy. This is the first example of the use of a mouse skull AIE point through the complete stroke visualizing process, and has a high penetration depth and good image contrast. Efficient emitters of these good results are expected to develop a noninvasive living brain biological imaging, having a strong nonlinear optical effect opens a new way. 首创AIE点无创3PF显微成像技术,实现活体脑卒中过程可视化

Photo Introduction

Molecular Design and Performance 1.AIE luminescent agents

Study design AIE luminescent agent BTF, having strong electron donor triphenylamine (TPA), t-butyl (t-Bu) group and an electron acceptor fumaronitrile (FN). BTF has a donor – acceptor (D-A) structure, having a molecular FR / NIR and visible emission multiphoton absorption. In addition, the BTF further comprising a benzene ring group and t-Bu more freely rotatably, conducive excited state energy consumption in solution form by intramolecular motion. These movements are limited aggregation, which makes it possible to exciton radiative decay process to facilitate the AIE. Furthermore, TPA twisted portions and BTF large t-Bu group hindered strong π-π stacking interactions formed. These factors make BTF having a long wavelength emission and high quantum efficiency. BTF has good solubility in common organic solvents, but insoluble in water. To study the effect of solvent polarity and aggregation BTF launch process, they measure different water content (FW) in THF / water mixture PL spectrum. BTF faint fluorescence (Figure 1C) in absolute THF. When a small amount of water (fw≤50%) in THF BTF, since a typical twisted intramolecular charge transfer (TICT) effect, reduced emission of BTF. In a polar environment, these molecules undergo rapid molecular acceptor moiety within the electron transfer from the donor to the molecule D-A conformational transition from a coplanar structure gauche conformation. With further increase in water content (fw≥60%), BTF molecule is formed due to the hydrophobic effect of nanoaggregates. Nano-aggregates of hydrophobic environment eased TICT effect, as the dominant factor in the AIE effect more pronounced, resulting in increased PL intensity BTF mixture. When fw = 90%, PL intensity maximum was 5-fold (FIG. 1D) absolute THF solution. Thus, BTF AIE is active molecule.BTF absolute THF solution fluorescence quantum efficiency ([phi] f) 2.7%, significant increase in the solid state to 42.6%.

FIG. 1 A) BTF chemical structure and B) synthetic routes. C) different water fraction (FW) in tetrahydrofuran / water mixture BTF PL spectrum. D) graph with the BTF THF / water mixtures the relative PL intensity (I / I0). Illustration: BTF powder fluorescence photograph at 365nm ultraviolet irradiation.

2. Biological imaging applications

FR aggregation body having a high ΦF of BTF / NIR emission, can be used for biological imaging. For the hydrophobic BTF has good water dispersibility in an aqueous medium, using amphiphilic polymer Pluronic F-127 was prepared as a biocompatible encapsulant BTF point (FIG. 2A). In an aqueous solution, a lipophilic moiety, and Pluronic F-127 AIE core molecules forming nano dots, a hydrophilic portion of the housing is formed into the water. BTF point of maximum absorption wavelength in aqueous solution is 500nm, 515nm laser excitation and business is very consistent. BTF point PL spectrum (645nm) at peak in the red region, and extends well into the near-infrared region (800nm). BTF point than traditional organic dyes (usually 130nm), which facilitates high-contrast imaging of biological applications, since smaller emission quenching tendency of self-absorption. BTF fluorescence quantum yield point ([phi] f) was 36.1%. BTF point long-term stability. Even if they are suspended in phosphate buffered saline solution (PBS) is stored for a long time at room temperature, the fluorescence spectra are almost unchanged. BTF cytotoxicity and low points. In different concentrations (12.5,25,50 and 100mg mL -1 ) and the HeLa cells after 24 hours of incubation, cell viability was maintained above 95%. BTF point having high chemical stability. The BTF point dispersing buffer solution at different pH values, significant change is not detected in the UV and PL. They continue to study the nonlinear optical properties of BTF points. (Fs) laser excitation, three 1550nm photons are absorbed simultaneously during the excitation of near-infrared femtosecond 1550nm, subsequent radiation of the same single-photon attenuation processProcess (FIG. 2B). Observed at 650 nm to about 3PF BTF bright point, a sharp signal peak was observed (FIG. 2B, C) from the third harmonic generation (THG) at 517 nm at. 3PF and THG are higher-order nonlinear optical effects. 3PF maximum emission wavelength is FR / NIR region of 650nm, it has a higher penetration depth and low light absorption, so the strong point of the BTF 3PF more suitable for imaging living organisms. At 1550nm, the three-photon absorption cross section σ3 BTF is 2.56 × 10 -79 cm 6 s 2 , much higher than the conventional organic dye Rh6G ( 6 × 10 -81 cm 6 s 2 ) Some dyes and previously reported. The higher the value of σ3 BTF point in favor of deep-tissue bioimaging.

FIG. 2 A) used as illustrated BTF point of the polymer matrix formation process F127. B) 1PF, and THG schematic 3PF process. C) 1550nm femtosecond laser excitation at BTF nonlinear optical properties of the aqueous dispersion. Illustration: THG and the capillary 3PF image BTF aqueous dispersion. D) 1550nm relationship femtosecond laser excitation and fluorescence intensity at point BTF power. E) 1550nm CW laser irradiation intensity at different points 3PF BTF time point.

First, they studied the use of imaging system 3PF mouse brain vessel through a small cranial window real-time imaging. As shown in FIG. 3A-C, and THG 3PF signal match well, suggesting that they are from the same nanoprobe. 3PF binding THG imaging mode and improves the reliability of deep tissue imaging. FIG. 3D-F show high-resolution three-dimensional images of different revascularization mouse brain penetration depth of from 0 to 900μm. Penetration through different combined image (FIG. 3J) depth, high-resolution three-dimensional image reconstructed from a different perspective (FIG. 3K, L), it provides a clear image on the spatial network of blood vessels and micro capillaries major details.

FIG. 3 is a penetration depth of 55μCerebrovascular m mice A) THG, B) 3PF, and C) the superimposed image. D-F) mouse brain vascular penetration depth from 0 to 900μm of the body three-dimensional high-resolution image. G, H) along a cross-sectional intensity profile (D) and (E), measured in the blue line. I) fluorescence decay curve. J) cerebrovascular fluorescence image of a different penetration depth. Mouse brain blood vessel penetration depth from 0 to 900μm of the 3D image K) side and L) a top view. Cerebrovascular mice

They further applied to a fluorescent BTF point skull complete visualization. Figures 4A-C show a three-dimensional high-resolution image reconstruction cerebrovascular mice, which provides a clear picture image of the main space and the vascular system of fine capillary network. FIG microcapillaries FWHM calculated 200μm depth of 0.95 m, as calculated at 1.59μm depth 300μm, calculated as 2.08μm (FIG. 5D-F) at a depth of 400μm. Cerebral thrombosis is a common disease of the brain that can lead to acute brain damage and even death. Thus, they are further utilized BTF nanodots monitored during a complete stroke skull mice. AIE point of first use of complete mouse brains 3PF skull imaging using infrared antireflection low magnification objective general information (FIG. 4G, H) vascular structures large field of view. In the normal state the brain can effectively monitor the signal strength of 92.1 3PF from the blood vessel. After cerebral thrombosis, 3PF strength decreased significantly low value to 1.1, indistinguishable from the background signal. AIE bright point based on a significant non-linear optical effects, a high contrast to their first noninvasively complete cerebral skull formation in live mice (FIG. 4L) were observed. High-resolution three-dimensional image of the living body

FIG. 4 A-C) in mouse brain vessel having a complete skull, the penetration depth of 0 to 400μm. D-F) in the (A) – a cross-sectional intensity profile (C), yellow line measurements. 3PF front image (G, H) and after (I, J) cerebrovascular G-J) cerebral thrombosis. K) in (G) and (I) is a cross-sectional intensity profile of the yellow line measurements. L) 3PF imaging microscopy using, cerebral thrombosis schematic process visualization based on the complete skull AIE point.

Highlights Summary

In summary, co-authorAIE became bright luminescent agent BTF, a powder having a quantum efficiency of up to 42.6%. The resulting AIE point having high brightness, large Stokes shift, good biocompatibility, good light stability and a large three-photon absorption cross section. They can serve as efficient fluorescent probe for penetration depth and high image contrast in vivo near infrared laser excitation of cerebral angiography. First reported by AIE point 3PF noninvasive imaging microscopy to observe the process in mice with stroke. These good results will help to develop highly efficient near-infrared solid-state transmitter for non-invasive monitoring of brain diseases or disorders. The full text link: https: //onlinelibrary.wiley.com/doi/10.1002/adma.202000364