Diagnosis of fatty liver disease by a multiphoton-active and lipid-droplet-specific AIEgen with nonaromatic rotors

Hojeong Park ab, Shijie Li c, Guangle Niu abd, Haoke Zhang a, Zhuoyue Song c, Qing Lu d, Jun Zhang a, Chao Ma e, Ryan T. K. Kwok ab, Jacky W. Y. Lam ab, Kam Sing Wong e, Xiaoqiang Yu d, Qingping Xiong *cf and Ben Zhong Tang *abg
aDepartment of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Institute for Advanced Study, Institute of Molecular Functional Materials and Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong 999077, China. E-mail: tangbenz@ust.hk
bHKUST-Shenzhen Research Institute, No. 9 Yuexing 1st RD, South Area, Hi-tech Park, Nanshan, Shenzhen 518057, China
cClinical Medical College of Acupuncture Moxibustion and Rehabilitation, Mathematical Engineering Academy of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China. E-mail: qpxiong@gmail.com
dCenter of Bio and Micro/Nano Functional Materials, State Key Laboratory of Crystal Materials, Shandong University, Jinan 250100, China
eDepartment of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong 999077, China
fJiangsu Key Laboratory of Regional Resource Exploitation and Medicinal Research, Faculty of Chemical Engineering, Huaiyin Institute of Technology, Huai'an 223003, Jiangsu, China
gCenter for Aggregation-Induced Emission, SCUT-HKUST Joint Research Institute, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China

Received 28th October 2020 , Accepted 23rd December 2020

First published on 23rd December 2020


Fatty liver disease (FLD) has become an increasing global health risk. However, an accurate diagnosis of FLD at an early stage remains a great challenge due to the lack of suitable imaging tools. To this end, we developed the fluorescent two-photon aggregation-induced emission (AIE) luminogen ABCXF for high-contrast imaging of FLD tissue. ABCXF has a large Stokes shift, good two-photon absorption cross-section, bright red emission, and high fluorescence quantum yield in the solid-state, and excellent photostability. It shows a planar intramolecular charge transfer (PICT) effect instead of a twisted intramolecular charge transfer effect in polar solvents. Photophysical and crystal data demonstrated that the nonaromatic rotors – trifluoromethyl (–CF3) contribute to its AIE effect. Biocompatible lipid droplet-targeting ABCXF can selectively light up lesions in the FLD tissue with a high signal-to-noise ratio. It shows better imaging performance compared to Oil Red O. Thus, ABCXF can be a potential alternative to Oil Red O for the diagnosis and evaluation of FLD from a liver biopsy.


At present, human health is highly threatened by various kinds of diseases. Hepatic steatosis, commonly known as fatty liver disease (FLD), is a medical condition where excess lipid droplets accumulate in hepatocytes in the form of triglyceride.1,2 It is categorized into two types: non-alcoholic fatty liver disease and alcoholic fatty liver disease.3 Recently, non-alcoholic FLD has received considerable attention as the prevalence of non-alcoholic FLD has increased to about 25% of the global population while no approved medication is available for the disease in the market.4 Non-alcoholic FLD is known to be strongly associated with characteristics of metabolic syndromes, such as obesity, type-2 diabetes, dyslipidaemia and hypertension.1,2,5 An early stage of non-alcoholic FLD is reversible and managed through adjustment in diet and physical activities before progressing into more severe advanced stages including cirrhosis and hepatocellular carcinoma.6 Therefore, early diagnosis of FLD with a reliable detection method is of paramount importance to the patients’ prognostic outcome.

Several imaging-based methods have been developed for the diagnosis of FLD including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasonography. However, CT requires a high level of exposure to X-ray radiation and MRI may be problematic for patients with uneven fatty change as it only allows them to evaluate a small sample volume.7,8 The major downfall of ultrasound is its high technical failure rate in morbidly obese patients.9 All these methods also suffer from low detection sensitivity. On the other hand, the ratio of the level of alanine transaminase (ALT) and aspartate transaminase (AST), which are important in liver function, in the blood may aid the diagnosis of FLD. Yet, as over 80% of the biochemistry remains normal, blood tests based on liver enzyme detection probably lead to patients remaining undiagnosed and untreated.10,11 Thus, a simple and reliable detection technique for the examination of the liver condition is in great demand.

In the modern clinical setting, diazo dyes such as Oil Red O are widely used for the diagnosis of FLD by histological staining of lipid droplets (LDs).12 Unfortunately, the performance is rather unreliable. Apart from their unstable chemical structures and lack of selectivity toward LDs, the sample preparation using these dyes is extremely time-consuming, and the use of organic solvents like ethanol and isopropanol during the staining procedure due to their poor solubility may cause artificial fusion of LDs.13,14 Furthermore, the shallow penetration in tissue fails to accurately visualize LDs via the conventional light microscopy.15 Recently, fluorescence microscopy has been widely utilized for monitoring cell physiology and medical diagnosis due to its high sensitivity, selectivity, non-invasiveness, and ability to provide excellent spatial and temporal resolution.16–18 Two commercially available probes Nile Red and BODIPY493/503 are commonly used for LD staining.19 However, Nile Red suffers from high unspecific background fluorescence signal and broad emission peak which hinders its use in multi-color imaging.20 BODIPY493/503 exhibits small Stokes shift to cause non-radiative energy loss and interference from the scattered light.21 Although some attempts have been made to develop new fluorescent probes for selective imaging of LDs,22,23 fluorescent probes have been rarely utilized in the diagnosis of FLD due to their unsatisfying permeability, limited tissue penetration, and poor signal-to-noise ratio.24,25 Generally, most of these reported probes usually work at low concentrations but their photostability by continuous irradiation especially high-intensity two-photon lasers is of concern. On the other hand, increasing the concentration of the hydrophobic traditional dyes leads to unfavorable aggregation-caused quenching (ACQ) phenomenon.26 Therefore, it is necessary and meaningful to develop ACQ-free and highly photostable fluorescent LD-specific probes for the diagnosis of FLD.

Recently luminogens with aggregation-induced emission (AIE) characteristics have received considerable attention due to their unique photophysical properties.27 AIE luminogens (AIEgen) are non-emissive or weekly emissive in solution but emissive upon aggregation or in the solid-state through a mechanism of the restriction of intramolecular motions (RIM).28,29 Based on RIM, numerous AIEgens have been developed for various biological applications such as fluorescence imaging, biosensing, and disease diagnosis.30–32 Compared with one-photon imaging, multiphoton like two-photon imaging using near-infrared excitation is more suitable in biomedical imaging because of the minimal background autofluorescence, low photobleaching, high spatial resolution, and deep tissue penetration.33–36 Considering their high fluorescence brightness and photostability, AIEgens are naturally stable and promising candidates for two-photon imaging.37,38 Since liver tissue suffers from autofluorescence covering the whole visible light region, it would be ideal to develop a novel multiphoton-active AIEgen.39,40 Conventionally, the introduction of an electron donor–acceptor (D–A) group into a π-conjugated system is a frequently used strategy to construct two-photon active fluorescent dyes.41 Such a strategy generally results in intramolecular charge transfer (ICT), which is beneficial for enhancing the electron mobility and red-shifted and intensity-increased fluorescence. However, most AIEgens show twisted intramolecular charge transfer (TICT) properties rather than an ICT effect, due to their highly twisted structures.42,43 Pure TICT effect is detrimental for bioimaging because of the highly polar water environment in biological systems.44,45 Therefore, the key to developing planar intramolecular transfer charge (PICT)-based AIEgens is to inhibit the TICT effect in a D–A system by constructing a planar core.46 As the highly twisted AIEgens with numerous aromatic rings and rotors have flexible cores, seeking some pendent nonaromatic rotors on the fluorescent planar cores will probably lead to novel D–A based AIEgens with favorable PICT effect.

Recently, a few potential bulky nonaromatic rotors have been reported.47,48 Panigati et al. developed a TMS (Me3Si) group substituted dinuclear rhenium complex with bright solid-state emission with a fluorescence quantum yield of over 50%, while it showed faint emission (fluorescence quantum yield of 6%) in toluene.47 The authors concluded that the intramolecular motions of the nonaromatic rotor of the TMS group quench the emission in solution and the restricted rotation of such a nonaromatic rotor is most likely responsible for the emission in crystals. In our previous work, we observed a phenomenon of one trifluoroacetyl group replaced compound N-((2-((9H-fluoren-9-ylidene)(phenyl)methyl)phenyl)-2,2,2-trifluoroacetamide) with no emission in solution or the aggregated state, which was completely AIE inactive.48 However, another acetyl group substituted compound N-(2-((9H-fluoren-9-ylidene)(phenyl)methyl)phenyl)acetamide was AIE active. We noticed that the trifluoromethyl group (CF3) is also a nonaromatic rotor ascribed to the lack of emission of compound N-((2-((9H-fluoren-9-ylidene)(phenyl)methyl)phenyl)-2,2,2-trifluoroacetamide).

Based on this, we anticipated that the introduction of the nonaromatic rotor CF3 group could be a promising design strategy to maintain the good π-conjugation of AIEgens as well as favorable PICT effect. With these considerations in mind, we designed and synthesized a new donor and acceptor incorporated fluorescent two-photon compound ABCXF by decorating the nonaromatic rotor (CF3) on the conjugated core. As we expected, single-crystal data indicate the planar structure of ABCXF. ABCXF is AIE-active and brightly red-emissive in the solid-state. The PICT effect of this AIEgen was verified by investigating the fluorescence data in different polar solvents. Combined photophysical and crystal data revealed that nonaromatic rotors (CF3) in ABCXF contribute to its AIE effect. Given its lipophilic structure, in vitro imaging data confirmed the high specificity of ABCXF in LDs. Compared with most LD dyes with complicated synthesis procedures, ABCXF was obtained via a one-step synthesis. It was further utilized for the diagnosis of FLD obtained from guinea pig FLD tissues by one-photon and two-photon microscopy and the photostability of ABCXF under continuous one- and two-photon irradiation was investigated.

Results and discussion

The synthesis route of ABCXF is depicted in Fig. 1A. ABCXF was obtained through a simple one-step nucleophilic reaction. Commercially available compound 1 and compound 2 were reacted in anhydrous EtOH in the presence of t-BuONa at 90 °C to yield ABCXF. ABCXF was characterized by 1H NMR, 13C NMR, 19F NMR, and HRMS spectroscopy (Fig. S1–S4, ESI). A detailed synthetic procedure was provided in the ESI. The structure of ABCXF was confirmed by the single-crystal structure analysis (Fig. 1B). It clearly shows that ABCXF almost exhibits a planar structure with a very small twisting angle of 10.55° between the trifluoromethyl substituted benzene plane and the other π-conjugated part of the molecule. A single crystal suitable for single-crystal analysis was obtained by slow evaporation of the solvent mixture of CH2Cl2 and MeOH (3[thin space (1/6-em)]:[thin space (1/6-em)]1, v/v) at room temperature. The details of the X-ray experimental conditions, cell data, and refinement data of ABCXF are summarized in Table S1 (ESI). Additionally, DFT calculations were performed with the method of b3lyp/6-31g(d,p) by using the Gaussian 09 program package (Fig. 1C). The highest occupied molecular orbital (HOMO) was mostly delocalized on the other part of the molecule except for the trifluoromethylphenzene moiety, whereas the lowest unoccupied molecular orbital (LUMO) was delocalized on the trifluoromethylphenyl substituted acrylonitrile unit. The energy bandgap between the HOMO and LUMO was 2.92 eV. The D–A based ABCXF showed typical intermolecular charge transfer (ICT) characteristics as we expected.
image file: d0qm00877j-f1.tif
Fig. 1 (A) Synthetic route of ABCXF. (B) ORTEP drawing of the single crystal structure of ABCXF with atoms labelled in color in different directions. CCDC 1904764. C, gray; H, white; F, green; N, blue. (C) Frontier molecular orbitals of ABCXF calculated, HOMO: highest occupied molecular orbital, LUMO: lowest unoccupied molecular orbital.

We systematically investigated its photophysical data. As shown in Fig. S5 (ESI), ABCXF possesses an absorption maximum (λabs) at 448 nm in THF which falls in the range of visible light allowing less photodamage to the biological system compared to UV light. The AIE property of ABCXF was studied in a THF/water mixture with different water fractions (fw) (Fig. 2A, B, and Fig. S6, ESI). ABCXF shows an emission maximum (λem) at 579 nm in THF solution and a large Stokes shift of 131 nm. With the addition of water to the THF solution, its emission showed a redshift and its intensity slightly increased as it reached fw = 70%. At fw = 95%, ABCXF showed the highest FL emission at 585 nm because of the formation of aggregates, indicating the aggregation-induced enhanced emission (AIEE) property. A shorter peak at about 605 nm also exists, due to different intermolecular interactions between ABCXF molecules in nanoaggregates. As the water fraction further increased to 99%, the FL intensity drastically decreased. Such a phenomenon has been frequently reported and explained due to a change in morphology and size of nanoparticles at high water fraction.28,49 It is noteworthy that the ratio of FL increase at fw = 95% compared to 0% is only 10 fold (Fig. 2B), possibly indicating the formation of loose packing aggregates. The light dynamic scattering measurements show the presence of nanoaggregates in fw = 95% and 99% with the size of about 153 nm and 143 nm in diameter, respectively (Fig. 2C and Fig. S7, ESI). ABCXF emits red-shifted emission (647 nm) in the solid-state compared with that in solution (Fig. 2D). Before analyzing the intermolecular packing mode of ABCXF in the crystal to explain the red-shifted solid-state emission, we first recorded the X-ray diffraction (XRD) pattern of a pristine solid sample. As shown in Fig. S8 (ESI), its XRD pattern was very similar to the simulated XRD pattern from X-ray single crystal data, indicating the easy crystallization of ABCXF and similar interactions in pristine samples. Then, the intermolecular packing mode of ABCXF in the crystal was analyzed (Fig. S9, ESI). It showed some π⋯π interactions between the electron-donating and withdrawing groups, resulting in strong intermolecular D–A interactions and red-shifted emission in the solid-state. Generally, previously developed AIEgens possess highly twisted structures due to the presence of multiple aromatic rotors, and restriction of the movement of these rotors is known to be the cause of AIE phenomena. However, in the case of ABCXF, except for intermolecular C–H⋯π interactions, multiple intermolecular C–H⋯F interactions between adjacent molecules are also formed, indicating the motion inhibition of the phenyl units and the CF3 group. In addition, the disordered assembly of the CF3 group attached to the phenyl could be observed (Fig. S10, ESI),50 further confirming the free motion of CF3 groups in the solution state but inhibited motion by the crystal lattice in the solid-state. The restricted molecular motion of the nonaromatic rotor (CF3 group) together with the aromatic rotors like the phenyl group contributes to preventing the loss of excited-state energy via nonradiative decay channels, resulting in bright emission in the solid-state. After strong grinding of the solid ABCXF, the fluorescence showed only a slight blue shift with a maximum wavelength of 625 nm (Fig. S11, ESI), indicating stable intermolecular interactions. The absolute fluorescence quantum yield (QY) of ABCXF in the solid-state and diluted THF solution was measured using an integrating sphere. In the diluted THF solution, the fluorescence QY of ABCXF is only 0.8% as the active intramolecular motion consumed the energy of the excited state through non-radiative processes. However, in the solid-state, ABCXF is highly emissive with the fluorescence QY of 15.9% due to the restriction of intramolecular motion (Fig. 2D).

image file: d0qm00877j-f2.tif
Fig. 2 (A) FL spectra of ABCXF (10 μM) in THF/water mixtures with different water fractions (fw). (B) The plot of FL emission intensity versus the composition of the THF/water mixture containing ABCXF. Inset: Fluorescent photographs of ABCXF in THF/water mixtures with different water fractions under 365 nm UV irradiation. (C) Dynamic light scattering data of ABCXF in water containing 5% THF. Hydrated diameter: 154 nm. (D) Normalized FL spectra of amorphous ABCXF solid. Inset: Fluorescent photograph of ABCXF taken under 365 nm UV irradiation.

As ABCXF is a donor–acceptor conjugated π-system, it is anticipated to display distinct fluorescence changes in different polar solvents. The λem of ABCXF gradually red-shifted from 550 to 620 nm with an increase in fluorescence intensity as the solvent polarity increased from toluene to DMSO (Fig. 3A). The fluorescence QYs of ABCXF in different solvents were also recorded using an integrating sphere (Table S2, ESI). These data together with the planar structure indicated that ABCXF shows positive solvatochromism in different polar solvents and enhanced emissions in highly polar solvents due to the excited-state charge transfer enhancement.51–53 TD-DFT calculations were performed to theoretically verify the solvatochromic behavior of ABCXF in different solvents. Based on the calculation result, we observed that, as the solvent polarity increases, the energy gap at the excited state narrows whereas the oscillator strength increases (Fig. S12 and Table S3, ESI). We further studied the effect of viscosity on ABCXF in glycerol/MeOH mixture with different fractions of glycerol. As shown in Fig. 3B, the fluorescence enhancement was observed as the viscosity of the solvent increased, due to the restriction of the intramolecular motion of ABCXF. Given the lipophilicity of ABCXF with a calculated log[thin space (1/6-em)]P (n-octanol/water partition coefficient) value of 5.842, we anticipated that ABCXF probably is an LD-specific dye.54 Therefore, we studied the photophysical property of ABCXF in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and trioleate glycerol (TAG) in PBS to mimic the environment of LDs. The chemical structures of DMPC and TAG are displayed in Fig. S13 (ESI). As shown in Fig. 3C, in the presence of key components of LDs, ABCXF displayed blue-shifted and enhanced fluorescence (about 3-fold) at about 525 nm compared to ABCXF only, which is beneficial for light-up fluorescence imaging in biological systems. By using the femtosecond pulse laser (800–1000 nm) excited fluorescence and rhodamine B as the standard,55 we measured the two-photon absorption (TPA) cross-section of ABCXF at different wavelengths. As displayed in Fig. 3D, ABCXF has the maximum TPA cross-section value of 180 GM at 850 nm. This result indicates that ABCXF is a good candidate to be utilized for two-photon fluorescence imaging.

image file: d0qm00877j-f3.tif
Fig. 3 (A) FL spectra of ABCXF (10 μM) in different polar solvents. (B) FL spectra of ABCXF (5 μM) in methanol and methanol/glycerol mixture with different glycerol fractions (fG). (C) FL spectra of ABCXF (1 μM) in PBS solution (pH = 7.2) with and without DMPC (40 μg mL−1) and TAG (80 μg mL−1). (D) Two-photon absorption (TPA) cross sections of ABCXF in THF. 1 GM = 10−50 cm4 s per photon.

Before evaluating its biological imaging applications, we first studied the cytotoxicity of ABCXF in HeLa and HepG2 cells by the standard MTT assay. Approximately 5000 cells were plated in each well of a 96 well plate and after the cells adhered to the plate surface, a medium containing a range of concentrations of ABCXF was added to each well. Over the range of concentrations from 0.5 to 10 μM, ABCXF shows negligible cytotoxicity in both HeLa and HepG2 cells (Fig. S14, ESI). Subsequently, live-cell imaging in HeLa and HepG2 cells was performed within the concentration range tested for the MTT assay by confocal laser scanning microscopy. After incubation of ABCXF at 200 nM for 20 min, spherical dots in HeLa and HepG2 cells could be visualized with a high signal-to-noise ratio of 21.27 (Fig. 4A and Fig. S15, ESI), probably indicating its location in LDs. However, the commercially available dye, Nile Red showed bright emission in LDs as well as non-specific fluorescence in the cytoplasm under the same conditions, resulting in unsatisfactory imaging contrast with the signal-to-noise ratio of 4.774. The in situ fluorescence spectrum of ABCXF in HeLa cells was acquired using the Lambda mode, and the data revealed that ABCXF exhibited blue-shifted in situ fluorescence (about 550–555 nm) in live cells due to its ICT characteristic and the low polar environment of LDs (Fig. S16, ESI). Considering the spectral overlap between ABCXF and Nile Red, we used another previously developed probe TTV for co-staining experiments.56 As shown in Fig. 4B, ABCXF showed good overlap with TTV with a Pearson's correlation coefficient (PCC) value of 0.898 and 0.808 in HeLa and HepG2 cells, respectively. These data demonstrated that our AIEgen ABCXF is a suitable LD probe that works at an ultra-low concentration for specific LD staining.

image file: d0qm00877j-f4.tif
Fig. 4 (A) Confocal laser scanning microscopy (λex = 488 nm for ABCXF and Nile Red) images of HeLa cells stained with ABCXF (200 nM) and Nile Red (200 nM). Scale bar: 10 μm. (B) Confocal laser scanning microscopy (λex = 488 nm for ABCXF and 405 nm for TTV) images of HeLa and HepG2 cells stained with ABCXF (200 nM) and TTV (2 μM). Scale bar: 10 μm.

As previous studies have shown that the hallmark of FLD is excess lipid accumulation in the liver tissue.1,57 ABXCF's ability to specifically stain LDs motivated us to investigate its ability to visualize LDs in lesions of the FLD tissue. We built an FLD model by providing a high-fat feeding diet to a group of male Hartley guinea pigs and as a control, a group of guinea pigs was fed with a normal diet under the same experimental conditions.58 The sectioned tissues were incubated with ABCXF for 1 h before the imaging. ABCXF showed fluorescence in the FLD tissue from the high-fat feeding guinea pig (Fig. 5A), due to its ICT effect and the viscous and low polar lipid environment (Fig. 3C). Only very faint emission was collected in the control group due to the low abundance of lipid droplets in the normal tissue. Due to spectral overlap of ABCXF, BODIPY 492/503, and Nile Red (Fig. S16, ESI), and TTV's failure to stain lipid droplets in liver tissue, we could not perform a co-stain experiment to confirm the staining location in lesions of the FLD tissue. Therefore, we stained the normal and high-fat feeding guinea pig liver tissues with Nile Red to confirm that the round droplets in the bright field are lipid droplets in FLD tissue (Fig. 5B). The data indicated that round droplets in the bright field are lipid droplets in FLD tissue. ABCXF displayed a lower fluorescence than Nile Red in normal feeding tissue (Fig. 5A and B). Compared to recently developed two fluorescent probes for the FLD tissue imaging with strong background fluorescence,22,59 ABCXF showed a highly improved signal-to-noise ratio, indicating its great potential in the diagnosis of FLD.

image file: d0qm00877j-f5.tif
Fig. 5 One-photon (λex = 488 nm) fluorescence images of normal and high-fat feeding guinea pig liver tissues stained with (A) ABCXF (1 μM) and (B) Nile Red (1 μM). Scale bar: 20 μm. (C) One-photon (λex = 488 nm) and two-photon (λex = 850 nm) fluorescence images of high-fat feeding guinea pig liver tissue stained with ABCXF (1 μM). Scale bar: 20 μm.

Compared with one-photon fluorescence imaging, two-photon fluorescence imaging with near-infrared excitation light is a promising imaging technique as the scattering coefficient is lower and the focus range is narrower, thus providing some outstanding advantages of deep tissue penetration, reduced photobleaching, and lower phototoxicity.60–63 Considering the good two-photon absorption cross-section of ABCXF, we carried out two-photon imaging of ABCXF in the FLD tissue. After incubation with ABCXF for 1 h, strong two-photon excited fluorescence signals in LDs were detected and showed good overlap with one-photon excited fluorescence signals (Fig. 5C). It should be noted that the two-photon fluorescence imaging displayed lower background fluorescence compared to the one-photon imaging.

One of the most outstanding merits of two-photon fluorescence imaging is that it enables deep penetration depth in tissue imaging. Considering the high-contrast two-photon imaging of ABCXF in lesions of 10 μm-thick FLD tissue, we investigated its penetration depth with a 100 μm-thick FLD tissue. The fluorescent images were captured every 3 μm along the z-axis. Under the excitation of 850 nm, the spherical LDs in the tissue sample were observed along the z-axis up to a depth of 42 μm (Fig. 6A), and the 3D two-photon fluorescence image was successfully constructed with a high resolution (Fig. 6B). However, compared to the two-photon imaging, the one-photon fluorescence imaging of ABCXF under 488 nm excitation only showed a shallow penetration depth of less than 30 μm along the z-axis (Fig. S17, ESI). These data demonstrate that ABCXF can be used for visualization of LDs in lesions of the FLD tissue.

image file: d0qm00877j-f6.tif
Fig. 6 (A) Two-photon (λex = 850 nm) fluorescence images of the high-fat feeding guinea pig liver tissue stained with ABCXF (2 μM) at different penetration depths. Scale bar: 20 μm. (B) Reconstructed 3D two-photon fluorescence images. (C) Bright-field and fluorescence images of the high-fat feeding guinea pig liver tissue stained with Oil Red O and ABCXF (1 μM), respectively, at different time points. Scale bar: 50 μm (pper) or 20 μm (lower). (D) Normalized color and fluorescence intensities obtained from images of the high-fat feeding guinea pig liver tissue stained with Oil Red O and ABCXF, respectively, at different time points.

Oil Red O has been used for visualization of lipid and fat deposits in cells and tissue over the past few decades under bright-field microscopy. In particular, Oil Red O staining has been widely used as a histochemical stain for quantifying liver steatosis in liver biopsy samples. However, the Oil Red O staining method suffers from low sensitivity and shallow tissue penetration compared to the fluorescence imaging method.15,64 Moreover, Oil Red O stained tissue must be analyzed within 24 h of staining due to its chemical instability and possible fusions of adjacent LDs.14 We stained the FLD tissue with Oil Red O and ABCXF separately to compare their imaging performances at the different time points of 0, 12, 24, and 48 h (Fig. 6C and D). After 48 h of staining, the color of Oil Red O was dramatically faded. However, the fluorescence intensity of ABCXF remained above 90% even after 48 h, demonstrating its better tissue imaging performance compared to Oil Red O for the diagnosis of FLD.

Encouraged by the desirable one- and two-photon imaging performance of ABCXF toward the visualization of LDs in lesions of the FLD tissue, we further studied its photostability under one-photon and two-photon excitations. First, we continuously irradiated HeLa cells stained with ABCXF (200 nM) with a 488-nm laser. Commercially available LD probes, Nile Red and BODIPY 493/503 were used as comparisons. As shown in Fig. 7A, after continuous irradiation for 20 min, more than 80% of the initial fluorescence of ABCXF intensity remained. Whereas, the fluorescence intensity of Nile Red and BODIPY 493/503 diminished to 59% and 22% of the initial fluorescence, respectively. We applied two-photon NIR excitation of 850 nm at the same continuous irradiation to further evaluate their photostability in the FLD tissue. It showed that ABCXF exhibits higher photobleaching resistance than Nile Red and BODIPY 493/503 (Fig. 7B). Previous studies demonstrated that the trifluoromethyl substituted fluorescent dyes tend to have higher photostability compared to those of unsubstituted ones as the trifluoromethyl group contributes to an increase in oxidation potential and decrease in electron density, thereby reducing the probability of the molecule being photobleached.65–67 This explains why ABCXF exhibits a higher photostability under continuous one-photon and two-photon irradiation.

image file: d0qm00877j-f7.tif
Fig. 7 (A) One-photon photostability of ABCXF, Nile Red and BODIPY 493/503 under continuous irradiation (λex = 488 nm for ABCXF, BODIPY 493/503 and Nile Red, laser power of 4%) in HeLa cells. (B) Two-photon photostability of ABCXF, Nile Red, and BODIPY 493/503 under the same continuous irradiation (λex = 850 nm) in the FLD tissues.


We successfully synthesized a novel AIEgen, ABCXF with unconventional nonaromatic rotors (CF3) through a simple one-step nucleophilic reaction. ABCXF possesses favorable characteristics including a large Stokes shift (131 nm), good two-photon absorption cross-section (180 GM at 880 nm), and bright red emission of 646 nm in the solid-state (fluorescence quantum yield of 15.9%). ABCXF showed a PICT effect, which was revealed by the red-shifted and intensity-enhanced fluorescence of ABCXF in polar solvents. In vitro cell imaging data confirmed the specific staining property of ABCXF in LDs. Due to its PICT effect and non-polar and viscous environment of LDs, ABCXF displayed blue-shifted and intensity-enhanced fluorescence in LDs and greatly increased emission in lesions of FLD tissue. As ABCXF is comprised of an electron-donor and acceptor in a conjugated π-system, it was utilized for two-photon fluorescence imaging with a high signal-to-noise ratio and tissue penetration depth of 42 μm. Moreover, ABCXF exhibits better performance compared to clinically used diazo dye, Oil Red O in terms of the staining procedure, sensitivity, penetration depth, and chemical stability. It also shows remarkably higher photobleaching resistance under continuous one- and two-photon irradiation compared to commercial LD probes, Nile Red and BODIPY 493/503. ABCXF could be a promising LD probe for the diagnosis of FLD. Our design strategy can potentially serve as a guide for the development of other new fluorescent systems with nonaromatic rotors and this work opens new avenues for PICT-based AIE systems to construct bright NIR-emissive fluorescent dyes for in vivo biomedical imaging.

Conflicts of interest

There are no conflicts to declare.


This work was partially supported by the National Basic Research Program of China (21788102), the University Grants Committee of Hong Kong (AoE/P-02/12), the Research Grants Council of Hong Kong (16305518, N_HKUST609/19 and C6009-17G), the Innovation and Technology Commission (ITC-CNERC14SC01 and ITCPD/17-9), the Science and Technology Plan of Shenzhen (JCYJ20170818113851132, JCYJ20170818113840164, and JCYJ20180507183832744), the Special Fund of Taishan Scholars Project of Shandong Province, China (tsqn201909012), the Opening Fund of Key Laboratory of Photochemical Conversion and Optoelectronic Materials, TIPC, CAS (PCOM202001), and the Program of Qilu Young Scholars of Shandong University.

Notes and references

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Electronic supplementary information (ESI) available: Materials and methods; synthetic details, NMR spectra and HRMS spectra; photophysical data; and imaging data. CCDC 1904764. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d0qm00877j
H. P., S. L. and G. N. contributed equally to this work.

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