Aggregation-Induced Emission Luminogen with Deep-Red Emission

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Aggregation-Induced Emission Luminogen with Deep-Red Emission for Through-Skull Three-Photon Fluorescence Imaging of Mouse Yalun Wang, Ming Chen, Nuernisha Alifu, Shiwu Li, Wei Qin, Anjun Qin, Ben Zhong Tang, and Jun Qian ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b05645 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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Aggregation-Induced Emission Luminogen with Deep-Red Emission for Through-Skull ThreePhoton Fluorescence Imaging of Mouse Yalun Wang1+, Ming Chen2+, Nuernisha Alifu1, Shiwu Li3, Wei Qin2, Anjun Qin3*, Ben Zhong Tang2*, Jun Qian1* 1

State Key Laboratory of Modern Optical Instrumentations, Center for Optical and

Electromagnetic Research, Joint Research Laboratory of Optics of Zhejiang Normal University and Zhejiang University, Zhejiang University, Hangzhou, 310058, China 2

Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research

Center for Tissue Restoration and Reconstruction, Division of Life Science, State Key Laboratory of Molecular Neuroscience, Institute for Advanced Study, Institute of Molecular Functional Materials, Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China 3

Guangdong Innovative Research Team State Key Laboratory of Luminescent Materials and

Devices, South China University of Technology, Guangzhou, 510640, China KEYWORDS: three-photon fluorescence microscopic (3PFM) imaging, aggregation-induced emission (AIE), through-skull, deep-tissue imaging, in vivo

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ABSTRACT: Imaging the brain with high integrity is of great importance to neuroscience and related applications. X-ray computed tomography (CT) and magnetic resonance imaging (MRI) are two clinically used modalities for deep-penetration brain imaging. However, their spatial resolution is quite limited. Two-photon fluorescence microscopic (2PFM) imaging with its femtosecond (fs) excitation wavelength in the traditional near-infrared (NIR) region (700-1000 nm) is able to realize deep-tissue and high-resolution brain imaging. However, it requires craniotomy and cranial window, or skull-thinning techniques, due to photon scattering of the excitation light. Herein, based on a type of aggregation-induced emission luminogen (AIEgen) DCDPP-2TPA with large three-photon absorption (3PA) cross-section at 1550 nm and deep-red emission, we realized through-skull three-photon fluorescence microscopic (3PFM) imaging of mouse cerebral vasculature without craniotomy and skull-thinning. Reduced photon scattering of 1550 nm fs excitation laser allowed it effectively penetrate skull and tightly focus onto DCDPP2TPA nanoparticles (NPs) in the cerebral vasculature, generating bright three-photon fluorescence (3PF) signals. In vivo 3PF images of the cerebral vasculature at various vertical depths were obtained, and a vivid 3D reconstruction of the vascular architecture beneath the skull was built. As deep as 300 µm beneath the skull, small blood vessels of 2.4 µm could still be recognized.

Brain imaging is vital to neuroscience and related clinical applications. Although X-ray computed tomography (CT) and magnetic resonance imaging (MRI) have been widely utilized for deep-penetration brain imaging, they are still limited due to low spatial resolution. Fluorescence imaging is promising for in vivo observations, due to high spatial resolution, free of radiation, abundant signals, and fast response.1 However, photon absorption and scattering of excitation or/and emission light in biological samples severely influence its penetration depth.2, 3


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Since the femtosecond (fs) excitation laser with its wavelength in the traditional near-infrared (NIR) region (700-1000 nm) has less photon absorption in bio-tissues, two-photon fluorescence microscopic (2PFM) imaging could provide much enhanced penetration depth. Nevertheless, 2PFM imaging still requires craniotomy and cranial windows, or skull-thinning techniques, due to distinct photon scattering of the excitation laser in the skull. It may destroy the integrity of cerebral vasculature, as well as give rise to inflammation on the brain tissue. Thus, it is better to adopt an imaging modality for brain activities study as intact as possible.4 Three-photon fluorescence microscopic (3PFM) imaging has been rapidly developed during past few years. Different from 2PFM imaging, the fs excitation wavelength of 3PFM imaging is usually in the NIR-II region (1000-1700 nm).5 Long-wavelength fs laser excitation with reduced photon scattering is able to effectively penetrate skull and tightly focus onto the biological tissues beneath, generating bright three-photon fluorescence (3PF) for deep-tissue imaging.6, 7 Thus, 3PFM imaging would be quite promising to observe brain structure and related activities with high integrity. Furthermore, in a multi-photon fluorescence (MPF) process, the fluorescence intensity falls off dramatically with the distance z from the focal plane, which is 1/z2 in two-photon fluorescence (2PF) process and 1/z4 in 3PF process, as 3PF is a higher-order nonlinear optical effect.8 Thus, in 3PFM imaging, the out-of-focus fluorescence could be greatly reduced, with its sectioning capability, spatial resolution and imaging contrast remarkably improved.9 Due to the lack of endogenous fluorophores in bio-tissues for 3PFM imaging, exogenous fluorophores are usually introduced to improve the imaging contrast.10 To date, various types of fluorescent probes have been developed. However, certain limitations still exist, including small three-photon absorption (3PA) cross-section (σ3) of fluorescent proteins,11, 12 potential toxicity


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and irregular blinking of inorganic quantum dots (QDs),13, 14 photo-thermal damage and excretion difficulty of metal nanoparticles,15, 16 as well as the aggregation-cause quenching (ACQ) problem of most commonly used organic dyes. Aggregation-induced emission (AIE) luminogen is a novel type of organic dye, and its fluorescence increases rather than quenches accompanied by the aggregation process.17 The mechanism of AIE can be explained by the theory of restriction of intramolecular motions (RIM), which blocks the non-radiative channel and opens up the radiative channel.18 Thus, by encapsulating AIE luminogens (AIEgens) within certain matrix to form nanoparticles (NPs), the intramolecular motions of AIE molecules can be suppressed, and the fluorescence quantum yield (η) of NPs is greatly improved.19 Meanwhile, the σ3 of AIE NPs can be facilely improved by increasing the amount of loaded AIEgens inside. Considering the 3PF efficiency of a fluorophore is determined by its three-photon action crosssection (ησ3), which is the product of 3PA cross-section and fluorescence quantum yield, good 3PF performance could be anticipated from AIE NPs with high loading amount of AIEgens.20 In addition, as each AIE NP is composed of a large amount of AIE molecules, when some of the AIE molecules were photo-bleached by light irradiation during imaging, the left unbleached ones could still give out fluorescence. Therefore, the photo-stability of AIE NPs is very good.21 Besides, AIE NPs are purely organic (AIEgens as the core and amphiphilic polymer as the matrix) without any inorganic component, making them quite biocompatible. AIE NPs with bright fluorescence, good photo-stability, excellent biocompatibility, as well as multiple surface functionality are quite appropriate for bioimaging.22, 23 So far, substantial AIEgens have been developed and utilized for one-photon confocal microscopic imaging and 2PFM imaging. However, not all the AIEgens have large value of σ3, especially at long-wavelength, and that’s why the reports on AIE NPs based in vivo 3PFM imaging are still very rare, not to mention


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through-skull 3PFM imaging of the mouse brain. Hence, it is necessary to design AIEgens with large σ3 at long-wavelength to fulfill 3PFM bioimaging and related applications.24, 25 In this paper, a deep-red emissive AIEgen 5,6-bis(4'-(diphenylamino)-[1,1'-biphenyl]-4yl)pyrazine-2,3- dicarbonitrile (DCDPP-2TPA) was synthesized, with large σ3. The AIE feature of DCDPP-2TPA was verified in the system of THF/water mixtures. Aqueously dispersible DCDPP-2TPA NPs were obtained through nanoprecipitation by using Pluronic F-127 (F127) as the encapsulation matrix.26 The chemical and optical features of DCDPP-2TPA NPs were characterized. Under the excitation of a 1550 nm fs laser, DCDPP-2TPA NPs exhibited bright 3PF signals with deep-red emission. The σ3 of DCDPP-2TPA was measured to be 2.95×10-79 cm6s2, much larger than that of most commonly used organic dyes, such as Rhodamine 6G (Rh6G, σ3 = 6×10-81 cm6s2).27 The photo-stability of DCDPP-2TPA NPs was very high, and the 3PF intensity decreased very little after long-time continuous scanning of 1550 nm fs laser excitation. Biocompatible DCDPP-2TPA NPs were then utilized for in vivo 3PFM imaging of the mouse cerebral vasculature. In the model of mouse with craniotomy, the penetration depth was as large as 785 µm. In the mouse with intact skull, thanks to the reduced photon scattering of 1550 nm fs laser excitation, a vivid 3D reconstruction of the vascular architecture was obtained. At a vertical depth of 300 µm beneath the skull, capillaries as small as 2.4 µm (Gaussian-fitted full width at half maximum, FWHM) could still be distinguished. In vivo 3PF lifetime imaging was also performed, and images with much better signal-to-noise ratio were obtained.

RESULTS AND DISCUSSION Molecular structure and AIE feature of DCDPP-2TPA. To achieve luminogens with distinct multi-photon absorption capability, the strategy of donor-acceptor (D-A) structure with large π-conjugation was always employed.28 Based on the reported AIEgen of 2,3-dicyano-5,6-


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diphenylpyrazine (DCDPP),29, 30 which shows strong electron-accepting ability, it is therefore reasonable to modify it with strong electron donor of triphenylamine (TPA) to create luminogen of DCDPP-2TPA with distinct electronic push-pull effect (Figure 1a). In addition, this design strategy endows DCDPP-2TPA with red emission property, which is beneficial to reduce signal loss in biological tissues during 3PFM imaging. Subsequently, 5,6-bis(4'-(diphenylamino)-[1,1'biphenyl]-4-yl)pyrazine-2,3- dicarbonitrile (DCDPP-2TPA, 5) was synthesized according to the synthetic routes in Scheme 1, whose structure was fully verified by 1H NMR spectroscopy, 13C NMR spectroscopy and high resolution mass spectra (HRMS). As shown in Figure S1-S6, satisfactory data corresponding to their structure were obtained. Compared with our previously reported red emissive AIEgen of 2,3-bis(4′-(diphenylamino)-[1,1′-biphenyl]-4-yl) fumaronitrile (TPATCN), DCDPP-2TPA can be prepared under a simpler condition, making the materials much easier to obtain.

Scheme 1. The synthetic route to AIEgen of DCDPP-2TPA (5).


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Figure 1. (a) Chemical structure of DCDPP-2TPA. Molecular weight: 769 Da. (b) Molecular conformation of DCDPP-2TPA optimized by DFT/B3LYP/6-31G(d,p). (c) Fluorescence spectra of DCDPP-2TPA in THF/water mixtures with various water fractions, fw = 0-90 vol%, 0.1 mgmL-1. (d) The corresponding fluorescence intensities of DCDPP-2TPA in (c). Excitation wavelength: 450 nm. The AIE behavior of DCDPP-2TPA was confirmed by measuring the fluorescence spectra of DCDPP-2TPA molecules in THF/water mixtures with different water fractions (fw) in volume (vol %). As shown in Figure 1c and 1d, with increasing fw in THF/water mixtures from 0% to 40%, the weak fluorescence of DCDPP-2TPA (around 690 nm) decreased gradually due to the twisted intramolecular charge transfer (TICT) effect, which has also been found in a series of other AIEgens.31, 32 Whereas, in mixtures with high fw from 50% to 90%, larger aggregates were formed, and RIM effect started to dominate.18 Meanwhile, the twisted conformation of DCDPP-


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2TPA (optimized by DFT/B3LYP/6-31G(d,p), Figure 1b) also prohibits the formation of π-π stacking. The collective effects determined a remarkably enhanced emission of DCDPP-2TPA around 640 nm in the aggregate state. Furthermore, it should be noted that non-aromatic double bonds were absent in DCDPP-2TPA molecules, making them rather stable even under highpower laser irradiation, compared to most existing red emissive AIEgens.33 Preparation and characterization of DCDPP-2TPA NPs. Aqueously dispersible DCDPP-2TPA NPs were obtained by encapsulating DCDPP-2TPA with amphiphilic polymer (F127) via a modified nanoprecipitation method (Figure 2a).26 The morphology of DCDPP2TPA NPs was visualized by a transmission electron microscope (TEM). DCDPP-2TPA NPs were found to have spherical appearances. By measuring the diameter of each DCDPP-2TPA NP in the TEM image (Figure 2b) and comparing it with the scale bar, the average diameter of NPs was calculated as ~29 nm. Through dynamic light scattering (DLS), the averaged hydrodynamic diameter (number-weighted) of DCDPP-2TPA NPs was further determined to be ~40 nm (Figure S7). Hereby, the size of DCDPP-2TPA NPs was very suitable for their circulation in blood vessels during bioimaging.34 Besides, extinction and one-photon fluorescence (1PF) spectra of the aqueous dispersion of DCDPP-2TPA NPs were also obtained. The absorption of DCDPP-2TPA NPs mainly located in the blue and violet region, with a peak around 440 nm (Figure 2c, blue line). Under daylight, it exhibited an orange appearance (insert of Figure 2c). The fluorescence spectrum of DCDPP2TPA NPs centered at 642 nm (Figure 2c). Under an ultraviolet (UV) lamp irradiation, DCDPP2TPA NPs gave out bright deep- red emission (insert of Figure 2c). Compared to other emission colors in the visible range, deep-red emission suffers less photon scattering by bio-tissues, thus providing better penetration depths.


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Figure 2. (a) Synthesis scheme of DCDPP-2TPA NPs, with F127 as the encapsulation matrix. (b) A TEM image of DCDPP-2TPA NPs. Scale bar: 100 nm. (c) Extinction spectrum (blue) and fluorescence spectrum (red) of DCDPP-2TPA NPs in aqueous dispersion. 0.03 mgmL-1 of DCDPP-2TPA. Excitation wavelength: 450 nm. Insert: photographs of DCDPP-2TPA NPs under daylight and under a UV lamp irradiation. (d) The nonlinear optical response of DCDPP-2TPA NPs in aqueous dispersion, under the 1550 nm fs laser excitation. Insert: the photograph showing the red 3PF signals in the aqueous dispersion of DCDPP-2TPA NPs. (e) The power dependence relationship of the fluorescence from DCDPP-2TPA NPs, under the same 1550 nm fs laser excitation. (f) The 3PF intensities of DCDPP-2TPA NPs, TPATCN NPs and TTF NPs at various time points, under continuous 1550 nm fs laser scanning. The quantum yield of DCDPP-2TPA NPs in aqueous dispersion was measured via a widely used comparative method.35 As shown in Figure S8a, the fluorescence intensity of DCDPP-2TPA NPs was in linear proportion to its concentration, indicating that the


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concentration range was proper and the self-absorption could be neglected. To minimize the deviations from the measurements, the fluorescence intensity of samples was averaged from many groups, including groups with various concentrations. The average fluorescence intensities of DCDPP-2TPA NPs and Rh6G (the reference standard) were subsequently obtained. According to equation (1) (in Methods), the quantum yield of DCDPP-2TPA NPs was calculated to be 5.7%. 3PF properties of DCDPP-2TPA NPs. Under the excitation of a 1550 nm fs laser (1 MHz), the nonlinear optical response of DCDPP-2TPA NPs was studied on a lab-built fluorescence measuring system (Figure S9). The 3PF spectrum of DCDPP-2TPA NPs was found to center at ~650 nm, with its tail extending into NIR range (Figure 2d). The deep-red/NIR emission was favored in deep-tissue bioimaging, because of the less photon attenuation in biotissues. Under the same 1550 nm fs laser excitation, the power dependence relationship of the fluorescence from DCDPP-2TPA NPs was then studied on the lab-built microscope system. For a kth order nonlinear process y = xk, we can get log y = k·log x through logarithm. As the same, the logarithm of the fluorescence intensity of DCDPP-2TPA NPs and the logarithm of the excitation power (Pin) were obtained, and they were found to have a good linear relationship (Figure 2e). The slope was calculated to be 2.84 (very close to 3), indicating that 3PF would be the main nonlinear optical process. Furthermore, under the excitation of a fs optical parametric amplifier (OPA, tuned to 1550 nm, 1 kHz), the σ3 of a DCDPP-2TPA molecule in chloroform was further measured on the lab-built system via the nonlinear transmissivity method (Figure S10).36 At 1550 nm, the σ3 of


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DCDPP-2TPA was measured to be 2.95×10-79 cm6s2, much larger than that of commonly used organic dye Rh6G (6×10-81 cm6s2).27 The bright 3PF signals from DCDPP-2TPA NPs would help to provide better signal-to-noise ratio during deep-tissue bioimaging. The lifetime of DCDPP-2TPA NPs was subsequently acquired via a time-correlated single-photon counting (TCSPC) system (Becker & Hickl GmbH, SPC-150), under the 1550 nm fs laser (1 MHz) excitation.37 As shown in Figure S11, according to the photon counting tracking of the 3PF signals from DCDPP-2TPA NPs, the lifetime was calculated to be 5.67 ns. As fluorescence lifetime is distinct for different fluorogens, lifetime imaging can help distinguish fluorescence signals from autofluorescence arising from biological tissues, achieving better signal-to-noise ratio. An in vivo 3PF lifetime image of the ear blood vessels in a mouse was demonstrated in Figure S12. As we can see, the mean 3PF lifetime of DCDPP-2TPA NPs inside the blood vessels was about 5.7 ns, and the contrast of lifetime image was very high. Chemical stability and photo-stability of DCDPP-2TPA NPs. The chemical stability of DCDPP-2TPA NPs was characterized by measuring their fluorescence intensity in aqueous dispersions with pH values of 1 to 13, phosphate buffer saline (PBS, 1×) and blood serum. As shown in Figure S13a, the fluorescence intensities of DCDPP-2TPA NPs in various dispersions (pH values of 1 to 13 and PBS) were almost the same at 0 hour. However, the fluorescence intensity in blood serum was only about the half of that in other solvents. This was due to the competition absorption of excitation light (450 nm) by blood serum, as demonstrated in Figure S13b. The time dependent stability of DCDPP-2TPA NPs was tested by further measuring their fluorescence in these dispersions at 24 hours and 2 weeks. In solutions with pH values of 1 to 11, PBS buffer and serum, the fluorescence intensities of DCDPP-2TPA NPs varied very little, while in solutions with pH values of 12 and 13, the fluorescence intensity of DCDPP-2TPA NPs


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dropped much. Thus, DCDPP-2TPA NPs had good chemical stability in acid (pH = 1-6), neutral (pH = 7), alkaline (pH = 8-11), saline (PBS 1×), and blood serum environments, but were not so stable in strong alkaline (pH = 12, 13) environment. The photo-stability of DCDPP-2TPA NPs was then characterized by measuring the 3PF intensities of DCDPP-2TPA NPs, TPATCN NPs, and 2,3-bis(4-(phenyl(4-(1,2,2triphenylvinyl)phenyl)amino) phenyl)fumaronitrile (TTF) NPs (as reference) dried on glass slides, under the continuous scanning of the 1550 nm fs laser (1 MHz). The 3PF images were showed in Figure S14, and the corresponding fluorescence intensities were calculated via integration. The time tracing of 3PF intensities was then acquired and plotted in Figure 3f. As we can see, upon irradiation, all of the AIE NPs were gradually bleached, and their 3PF intensities decreased correspondingly. After continuous scanning for 20 min, the 3PF intensity of DCDPP2TPA NPs lost about 20%, while the 3PF intensity of TPATCN NPs and TTF NPs lost about 30% and 40%. Considering that TTF NPs and TPATCN NPs are very stable,21, 38 the photo-stability of DCDPP-2TPA NPs was more advantageous. Different from the structures of TTF and TPATCN molecules, non-aromatic double bonds were absent in DCDPP-2TPA molecules (Figure S15). As non-aromatic double bonds are most susceptible to singlet oxygen, they are the main factor determining the rate of singlet oxygen induced degradation.39, 40 The absence of non-aromatic double bonds in DCDPP-2TPA molecules would help to improve the photo-stability of molecules. Those photo-stable DCDPP-2TPA NPs would be very suitable for long-term 3PF imaging. Histology analysis of DCDPP-2TPA NPs. To study the biocompatibility of DCDPP2TPA NPs, the commonly used histology analysis was applied. The mice in experimental group were injected with PBS (1×) dispersion of DCDPP-2TPA NPs, and the mice in control group


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were injected with only PBS (1×) solution. As shown in Figure S16 (imaged with a 40× objective lens) and Figure S17 (imaged with a 10× objective lens), 30 days post-injection of DCDPP-2TPA NPs, mice had no inflammation or abnormalities on their major organs (heart, liver, spleen, lung, kidney, and brain). In addition, the behavior of mice in the control group and experimental group was also studied. 30 days after the treatment, there were no apparent pathological differences in shape, weight, eating, drinking, exploratory behavior or activity between the two groups. These results demonstrated that the biocompatibility of DCDPP-2TPA NPs was very good.

Figure 3. In vivo 3PFM images of the brain blood vessels on a mouse with skull opened, at various vertical depths. (a)-(l) 0-785 µm. (m) and (n) 3D reconstructions of the blood vessels, at


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0-785 µm. (o) 3D reconstruction of the blood vessels, at 635-785 µm (the last 150 µm). Excitation: 1550 nm fs laser (1 MHz). Scale bar: 100 µm. In vivo 3PFM imaging of the brain vasculature on mouse with skull opened. In virtue of the bright 3PF signals from DCDPP-2TPA NPs, they were utilized for in vivo imaging of the brain blood vessels of mice. After the removal of skull in a small area, the mouse was injected with DCDPP-2TPA NPs and its head was imaged under the upright scanning microscope equipped with the 1550 nm fs laser (1 MHz). As shown in Figure 3a-3l, the 3PF images of brain blood vessels at various vertical depths were obtained, and the blood vessels could be distinguished till 785 µm. The corresponding 3D reconstructions were then built through these images (Figure 3m, 3n), showing the elegant brain vasculature vividly. Figure 3o showed the 3D reconstruction of blood vessels at the depth of 635 µm - 785µm (the last 150 µm). Even at such a deep penetration, the blood vessels could still be visualized clearly with good contrast. In vivo 3PFM imaging of the brain vasculature on mouse with intact skull. DCDPP2TPA NPs were further utilized for in vivo brain vasculature imaging of mice with intact skulls under the same 1550 nm fs laser excitation (Figure 4a). Through-skull brain imaging could keep the integrity of cerebral vasculature, as well as avoid inflammation on the brain tissue. 3PF images of the vertical sections of the brain blood vessels at various depths were obtained, and some typical results were showed in Figure 4b-4h. On the cranium layer, no 3PF signal was detected, indicating that there was no staining or accumulation of DCDPP-2TPA NPs in the skull. When the fs excitation laser with increasing power was focused onto the meninges layer, 3PF signals started to appear (Figure 4b). It would come from the DCDPP-2TPA NPs in the blood vessels of this layer, and this depth was then defined as 0 µm. When the imaging depth was larger than 100 µm, plenty of blood vessels could be observed (Figure 4d-4f). Within these


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depth ranges, various types of blood vessels, including the major blood vessels, the junctions and the capillaries could be visualized very clearly. To evaluate the imaging quality, a line was drawn across the capillary and the pixel intensities on it were plotted (Figure 4e, 4i). By Gaussian fitting, the full width at half maximum (FWHM) of the capillary was calculated to be 2.0 µm (Figure 4i),41 indicating that blood vessels as small as 2.0 µm could be recognized at 150 µm beneath the skull. Meanwhile, the signal-to-background ratio (SBR) was calculated to be as high as 20.4, which would ensure good imaging contrast. When further increasing the focusing depth, the quality of images began to deteriorate, mainly due to the photon scattering of skull and brain tissues (Figure 4f-4g). When the focusing depth reached ~300 µm, the influence of photon scattering became so distinct that only part of the blood vessels could be recognized (Figure 4h). By drawing a line across the blood vessel and plotting the intensity profile on it (Figure 4h, 4j), the Gaussian-fitted FWHM was found to be 2.4 µm, with a SBR of 7.1. Although the values were not as good as that obtained at the depth of 150 µm (beneath the skull), 3PFM imaging with DCDPP-2TPA NPs really still provided good performance on consideration of the strong photon scattering suffered from mouse skull. Thus, an in vivo penetration depth of 300 µm beneath the skull was obtained on the mouse with intact skull. Besides, the thickness of mouse skull was measured to be ~400 µm with a vernier caliper (Figure S18). Therefore, a total imaging depth of 700 µm was reached (from the surface of the skull). The bright 3PF signals from DCDPP-2TPA NPs, together with their good photo-stability and biocompatibility, contributed to the successful imaging of mouse brain. The deep penetration, high resolution and good contrast obtained here demonstrated the superiority of AIE NPs assisted 3PFM imaging for brain vasculature with intact skulls. The 3D reconstruction of the brain vasculature was subsequently built (Figure 4k). The major blood vessels and some small capillaries could be distinguished clearly. Video S1


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(Supporting Information) provided a vivid showing of the blood vessels at various vertical depths. More areas of the mouse brain were further observed with 3PFM imaging, and one of them was selectively showed in Figure 4l. Plenty of blood vessels could also be clearly visualized with high contrast. Through-skull 3PFM imaging has great meanings to non-invasive study of brain structure and functions, which requires high spatial resolution and certain penetration depth.

Figure 4. In vivo 3PFM imaging of the brain blood vessels on a mouse with intact skull. (a) A demonstration of the imaging area in the mouse brain. (b)-(h), 3PF images of the blood vessels at various vertical depths, 0-300 µm. (i)-(j) FWHMs of blood vessels and SBR analysis of 3PF


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images at 150 µm (i) and 300 µm (j). (k) 3D reconstruction of the blood vessels in mouse brain, 150-300 µm. (l) A 3PF image of the blood vessels at another area in mouse brain. Excitation: 1550 nm fs laser (1 MHz). Scale bar: 100 µm. Based on the distinguished 3PF lifetime of DCDPP-2TPA NPs (Figure S11), we further carried on the in vivo 3PF lifetime imaging of the brain blood vessels in the mouse. As shown in Figure 5a, the value of 3PF lifetime of DCDPP-2TPA NPs inside the blood vessels was around 5.8 ns, in good accordance to the measured lifetime of DCDPP-2TPA NPs (5.67 ns) in the glass capillary (Figure S11). In addition, the distribution of 3PF lifetime was quite uniform in the blood vessels. As the fluorescence lifetimes of different fluorogens were distinct and independent on the fluorescence intensity, lifetime images could provide much better signal-tonoise ratio.

Figure 5. In vivo functional imaging of the brain blood vessels on a mouse with intact skull. (a) A 3PF lifetime image of the blood vessels. (b) Measurement of the blood flow velocity (dx/dt). Excitation: 1550 nm fs laser (1 MHz). Scale bar: 100 µm.


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The brain blood flow velocity is relevant to various types of neural activities and it is an important indicator to monitor brain conditions. Here, the brain blood flow velocity was noninvasively measured with the through-skull 3PFM imaging. During the imaging process, as the intravenously injected DCDPP-2TPA NPs existed in the blood plasma, rather than stained on the red blood cells (RBCs), the RBCs would appear as shadows flowing with the fluorescent plasma. As shown in Figure 5b, a line scan along the capillary was used to detect the RBCs flow and its instantaneous velocity (dx/dt) was calculated to be ~2.4 mm/s. To be more intuitive, Video S2 (Supporting Information) recorded the live process of blood flow. The measurement of blood flow velocity would be helpful to detect the brain states.

CONCLUSION In summary, an AIEgen DCDPP-2TPA with large 3PA cross-section and deep-red emission was synthesized and characterized. Aqueously dispersible DCDPP-2TPA NPs were obtained through nanoprecipitation by using F127 as the encapsulation matrix. DCDPP-2TPA NPs had small size (~29 nm, TEM) and good water dispersibility. Under the excitation of a 1550 nm fs laser, DCDPP-2TPA NPs gave out bright 3PF emissions, with good photo-stability. Besides, DCDPP2TPA NPs showed good chemical stability and biocompatibility. Afterwards, upon the 1550 nm fs laser excitation, DCDPP-2TPA NPs were utilized as contrast agents for intravital 3PFM brain imaging of the mouse with and without the craniotomy model. Even for the mouse with an intact skull, a vivid 3D reconstruction of the brain vasculature could still be obtained. A penetration depth of 300 µm beneath the skull was achieved, and capillaries as small as 2.4 µm (FWHM) were recognizable. Deep-tissue, high-resolution, and through-skull 3PFM brain imaging provides a promising solution for non-invasive visualization of the cortex structure and functions.


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MATERIALS AND METHODS Materials and instruments. The aggreagtion-induced emission luminogen (AIEgen) 5,6-bis(4'-(diphenylamino)-[1,1'-biphenyl]-4-yl)pyrazine-2,3- dicarbonitrile (DCDPP-2TPA) was synthesized by ourself, and the details were described below. The AIEgen 2,3-bis(4-(phenyl(4(1,2,2-triphenylvinyl)phenyl)amino)phenyl)fumaronitrile (TTF) was prepared in Tang’s group.21 The AIEgen 2,3-bis(4′-(diphenylamino)-[1,1′-biphenyl]-4-yl) fumaronitrile (TPATCN) was provided by Lu’s group.28 Chloroform and ethanol were obtained from Sinopharm Chemical Reagent Co., Ltd. Pluronic F-127 (F127) and other chemical reagents were purchased from Alfa Aesar and Sigma Aldrich unless otherwise stated. Deionized (DI) water was used in all of the experiments. Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl under nitrogen immediately prior to use. 1

H and 13C NMR spectra were measured with a Bruker AVIII 400 spectrometer using

CDCl3 or CD2Cl2 as solvent. When CDCl3 was used, thetetramethylsilane (TMS; δ=0) was used as internal reference. High resolution mass spectra (HRMS) were recorded using a GCT premier CAB048 mass spectrometer operated in MALDI-TOF mode. The morphologies of DCDPP2TPA NPs were taken by a transmission electron microscope (TEM, JEM-1200EX, JEOL, Japan) operating at 160 kV on bright-field mode. Some TEM images were joined together to give an overall view. The hydrodynamic diameter of DCDPP-2TPA NPs were determined by dynamic light scattering (DLS) on a particle size analyzer (Malvern, Zetasizer Nano ZS-90) with an angle of 90 degrees. The extinction spectra were measured on a UV-vis scanning spectrophotometer (UV-2550, Shimadzu, Japan), and the fluorescence (FL) spectra were obtained on a lab-built fluorescence measuring system. Under the excitation of a 1550 nm fs laser (FLCPA-01C, Calmar


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Laser, 400 fs, 1 MHz), the lifetime of AIE NPs was measured via a time-correlated single-photon counting (TCSPC) system (Becker & Hickl GmbH, SPC-150).37 Synthesis of 1,2-bis(4'-(diphenylamino)-[1,1'-biphenyl]-4-yl)ethane-1,2-dione (3). Into a vacuum-evacuated and nitrogen-filled 500 mL two-necked round bottom flask was added 4,4’-Dibromobenzil (1, 2 g, 5.4mmol ), (4-(diphenylamino)phenyl)boronic acid (2, 3.92 g, 13.6 mmol), and Pd(PPh3)4 (314 mg, 0.27 mmol). After injection of 150 mL of distilled THF and 75 mL of K2CO3 aqueous solution (2 M), the reaction was kept for reflux at 80℃ for 8 hours. After that, the mixture was extracted with dichloromethane and washed by water for several times. The collected organic phase was then dried over anhydrous Na2SO4. After filtration and vacuum distillation, the crude product was purified by a silica gel column with dichloromethane/hexane (1:2 by volumn) as eluent. An orange powder of 1,2-bis(4'-(diphenylamino)-[1,1'-biphenyl]-4yl)ethane-1,2-dione was obtained in yield of 91.1%. 1H NMR (400MHz, CDCl3), δ (ppm): 8.06 (d, 4H), 7.72 (d, 4H), 7.53 (d, 4H), 7.31 (t, 8H), 7.16 (m, 12H), 7.10 (t, 4H). 13C NMR (100MHz, CDCl3), δ (ppm): 194.2, 148.6, 147. 3, 147.0, 132.5, 131.2, 130.6, 129.5, 128.1, 126.9, 125.0, 124.0, 123.0.HRMS (MALDI-TOF): m/z 696.2750 [M+, calcd for 696.2777]. Synthesis of 5,6-bis(4'-(diphenylamino)-[1,1'-biphenyl]-4-yl)pyrazine-2,3dicarbonitrile (DCDPP-2TPA, 5). Into a 100 mL round bottom flask was added 1,2-bis(4'(diphenylamino)-[1,1'-biphenyl]-4-yl)ethane-1,2-dione (3, 600 mg, 0.86 mmol), 2,3diaminomaleonitrile (4, 112 mg, 1.03 mmol) and 40 mL of acetic acid. The reaction was allowed for stirring and reflux at 130℃ for 8h. After that, the mixture was cooled down to the room temperature and poured into the ice water, followed by extracted with dichloromethane. The collected organic phase was washed by water for several times and then dried over anhydrous


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Na2SO4. After filtration and vacuum distillation, the crude product was purified by a silica gel column with dichloromethane/ hexane (1:2 by volumn) as eluent. A red powder of 5,6-bis(4'(diphenylamino)-[1,1'-biphenyl]-4-yl)pyrazine-2,3-dicarbonitrile (5) was obtained in yield of 75.5%. 1H NMR (400MHz, CD2Cl2), δ (ppm):7.68 (d, 4H), 7.61 (d, 4H), 7.52 (d, 4H), 7.30 (t, 8H), 7.12 (16H). 13C NMR (100MHz, CD2Cl2), δ (ppm): 154.9, 147.4, 143.2, 133.6, 132.6, 130.4, 129.4, 127.7, 126,6, 124,8, 123.4, 123.1, 113.5. HRMS (MALDI-TOF): m/z 768.2999 [M+, calcd for 768.3001]. Synthesis of AIE NPs. DCDPP-2TPA NPs were synthesized via a modified nanoprecipitation method,26 as shown in Figure 2a. Briefly, chloroform solution of DCDPP2TPA (200 µL, 1 mgmL-1) and chloroform solution of F127 (120 µL, 10 mgmL-1) were added into a flask. The obtained mixture was then dried under vacuum in a rotary evaporator to remove the chloroform completely. After that, DI water (200 µL) was added and the flask was under sonication for several minutes. Finally, the aqueous dispersion of DCDPP-2TPA NPs was obtained. Following the same procedure, TPATCN NPs and TTF NPs were also synthesized. Setup of the 1PF & 3PF measuring system. A lab-built fluorescence measuring system was applied to record the fluorescence spectra of AIE NPs under one-photon or three-photon excitations, as shown in Figure S9. For one-photon excitation, a 450 nm continuous wave (CW) laser was adopted. For three-photon excitation, a 1550 nm fs laser (FLCPA-01C, Calmar Laser, 400 fs, 1 MHz) was adopted unless specially declared. The laser beam from the light source was focused onto the sample in a cuvette by a lens (f = 25 mm). The focus was kept close to the edge of the cuvette to minimize self-absorption by the sample. The one-photon fluorescence (1PF) or three-photon fluorescence (3PF) signals from the sample were collected by an objective lens (XLUMPlanFLN, 20 ×, NA = 1.00), with a 90° angle to the beam propagation direction. After


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passing through the objective lens and the adjacent collimator, the fluorescence signal was then directed into a spectrometer (PG2000, Ideaoptics Instruments) through fiber. The data were finally recorded on the computer equipped to the spectrometer. Measurement of fluorescence quantum yield. The fluorescence quantum yield (η) of the aqueous dispersion of DCDPP-2TPA NPs was measured via the standard comparative method.35 Freshly prepared Rhodamine 6G (Rh6G) in DI water was selected as the reference.42 The extinction spectra of the reference and the sample were recorded on a UV-vis scanning spectrophotometer (UV-2550, Shimadzu, Japan), with their optical density (OD) below 0.1 to minimize the self-absorption of fluorescence. The fluorescence spectra of them were then acquired by the lab-built fluorescence measuring system (in Figure S9) under the same excitation. The fluorescence intensities were thus obtained through wavelength integration. Finally, the quantum yield of DCDPP-2TPA NPs was obtained as:35 A0 F1 n12 η1 = η0 A1 F0 n02

(1)

Where A is the extinction of samples, F is the average fluorescence intensity of samples from many groups (including groups of various concentrations) to minimize the deviations. The parameter n is the refractive index of the solvent (here both solvents are water, n1 = n0). The subscripts 0 and 1 represent the reference (Rh6G in water) and the sample (DCDPP-2TPA NPs in water). Measurement of 3PA cross-section. The three-photon absorption (3PA) cross-section (σ3) of DCDPP-2TPA molecules was measured via the classical nonlinear transmissivity method,36 as shown in Figure S10. To obtain high peak intensity, an optical parametric amplifier


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(OPA) system with low repetition (Libra-USP-HE, 160 fs, 1 kHz) was adopted as the excitation, and its output wavelength was tuned to 1550 nm. The laser beam was focused onto the cuvette containing the chloroform solution of DCDPP-2TPA molecules via a lens (f = 125 cm). The beam intensities in front of (I0) and at the back of the sample (I1) were measured by a power meter. The 3PA coefficient (in units of cm3GW-2) of DCDPP-2TPA molecules was obtained according to the following equation:36

γ=

1 1 ( 2 − 1) 2 2I 0 l0 T

(2)

Where I0 is the peak intensity of the incident light (in units of GWcm-2), l0 is the inner length of the cuvette (in units of cm), and T is the transmissivity of the material (I1/I0). The 3PA cross-section (in units of cm6GW-2) was further obtained as:36

σ3 ' =

γ N A d 0 × 10 −3

(3)

Where NA is the Avogadro constant and d0 is the molar concentration of DCDPP-2TPA molecules in chloroform (in units of cm-3). The 3PA cross-section (in units of cm6s2) was then defined as σ3 = σ3’(hν)2, where h is the Plank constant and ν is the frequency of the incident light. Animal preparation. All the animal experiments were conducted in accordance to the rules of Zhejiang University Animal Study Committee for the care and use of laboratory animals in research. The animals were fed with clean water and standard laboratory chow and their housing area were kept at 24°C with a 12 hour day/night cycle. Histology analysis of DCDPP-2TPA NPs. One group of mice were intravenously injected with phosphate buffer saline (PBS, 1×) dispersion of DCDPP-2TPA NPs (200 µL, 1


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mgmL-1) as the experimental group, and another group of mice were injected with only PBS (200 µL, 1×) solution as the control group. 30 days post-injection, both of the experimental group and the control group were sacrificed, and their major organs (brain, heart, liver, spleen, lung, and kidney) were resected through surgery. Tissue samples were then harvested and fixed in 4% paraformaldehyde overnight at 4 °C. After that, the tissue samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The histological sections were imaged under an inverted optical microscope with objective lenses of 40× and 10× magnifications at bright-field mode, respectively. In vivo 3PF imaging of the brain blood vessels of mice. 8-week-aged female BALB/c mice were used for in vivo brain vascular imaging. For through-skull imaging, the mice were anesthetized and their scalps were removed, while keeping their skulls intact. A smooth metal strip with a hole in the center was adhered to the mouse skull through dental cement. Then the metal strip was mounted onto a plate to immobilize the mouse during imaging. For open-skull imaging, the mice were anesthetized and both of their scalps and skulls were removed through microsurgery. A small smooth metal ring with a handle was then mounted onto the opened brain of each mouse. After that, a round thin cover glass slide was embedded in the ring and adhered to the mouse through dental cement to protect the brain, as well as offer a cranial window. The metal ring was then connected to a heavy metal plate to immobilize the mice during imaging. At last, the mice were intravenously injected with PBS (1×) dispersion of DCDPP-2TPA NPs (200 µL) via their tail veins prior to imaging. For 3PF imaging of the brain vasculature of mice, an upright scanning microscope (Olympus, BX61W1-FV1200) equipped with a 1550 nm fs laser (FLCPA-01C, Calmar Laser, 400 fs, 1 MHz) was adopted. The fs laser beam was focused onto the mouse brain by an


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objective lens (XLPlan N, 25×, NA = 1.05, work distance = 2.0 mm) with large NA and good near-infrared (NIR) transmissivity. The 3PF signals from DCDPP-2TPA NPs were then collected by the same objective lens. Passing through the dichroic mirror and a 590 nm long pass filter, the 3PF signals were collected by the photomultiplier tube (PMT) via non-descanned detection (NDD). 3PF images of brain blood vessels were taken at every 10 µm and the three-dimension (3D) reconstruction was further performed by software (Imaris).

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Synthesis and characterization of DCDPP-2TPA. Setup of 1PF & 3PF measurement. Setup of 3PA cross-section measurement. Further characterization of DCDPP-2TPA NPs. Histology study of DCDPP-2TPA NPs. Measurement of the skull thickness. (PDF) Throughskull 3PFM imaging of the mouse brain at various vertical depths. (AVI) Through-skull 3PFM imaging of the mouse brain at various time points. (AVI)

AUTHOR INFORMATION Corresponding Author *Jun Qian, E-mail: [email protected] * Ben Zhong Tang, E-mail: [email protected] * Anjun Qin, E-mail: [email protected] Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. + These authors contributed equally.

ACKNOWLEDGMENT This work was supported by National Basic Research Program of China (973 Program; 2013CB834701 and 2013CB834704), the Zhejiang Provincial Natural Science Foundation of China (LR17F050001), the National Hi-Tech Research and Development Program of China (863 Program; 2015AA020515), University Grants Committee of Hong Kong (AoE/P-03/08), the Research Grants Council of Hong Kong (16305015, 16308016, and N-HKUST604/14), the Innovation and Technology Commission (ITC-CNERC14SC01) and the Science and Technology Plan of Shenzhen (JCYJ20160229205601482). We thank Mr. J. W. Li for the help of pixel intensity extracting from 3PF images.

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TOC FIGURE

For Table of Contents Only A red emissive aggregation-induced emission luminogen (AIEgen) named DCDPP-2TPA was synthesized. DCDPP-2TPA NPs were obtained and they exhibited bright three-photon fluorescence (3PF) under the 1550 nm fs laser excitation. In vivo through-skull 3PF imaging of the brain vasculature was realized on DCDPP-2TPA stained mice, with a penetration depth of 300 µm beneath the skull.


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Aggregation-Induced Emission Luminogen with Deep-Red Emission

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