Spatiotemporal Visualization of Cell Membrane with Amphiphilic Aggregation-Induced Emission-Active Sensor

Open AccessCCS ChemistryRESEARCH ARTICLE1 May 2022Spatiotemporal Visualization of Cell Membrane with Amphiphilic Aggregation-Induced Emission-Active Sensor Youheng Zhang, Qi Wang, Zhirong Zhu, Weijun Zhao, Chenxu Yan, Zhenxing Liu, Ming Liu, Xiaolei Zhao, He Tian and Wei-Hong Zhu Youheng Zhang Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, Shanghai Key Laboratory of Functional Materials Chemistry, Institute of Fine Chemicals, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237 Google Scholar More articles by this author , Qi Wang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, Shanghai Key Laboratory of Functional Materials Chemistry, Institute of Fine Chemicals, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237 Google Scholar More articles by this author , Zhirong Zhu Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, Shanghai Key Laboratory of Functional Materials Chemistry, Institute of Fine Chemicals, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237 Google Scholar More articles by this author , Weijun Zhao Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, Shanghai Key Laboratory of Functional Materials Chemistry, Institute of Fine Chemicals, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237 Google Scholar More articles by this author , Chenxu Yan Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, Shanghai Key Laboratory of Functional Materials Chemistry, Institute of Fine Chemicals, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237 Google Scholar More articles by this author , Zhenxing Liu Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, Shanghai Key Laboratory of Functional Materials Chemistry, Institute of Fine Chemicals, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237 Google Scholar More articles by this author , Ming Liu Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, Shanghai Key Laboratory of Functional Materials Chemistry, Institute of Fine Chemicals, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237 Google Scholar More articles by this author , Xiaolei Zhao Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, Shanghai Key Laboratory of Functional Materials Chemistry, Institute of Fine Chemicals, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237 Google Scholar More articles by this author , He Tian Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, Shanghai Key Laboratory of Functional Materials Chemistry, Institute of Fine Chemicals, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237 Google Scholar More articles by this author and Wei-Hong Zhu *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, Shanghai Key Laboratory of Functional Materials Chemistry, Institute of Fine Chemicals, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai 200237 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202100967 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail High-fidelity spatiotemporal monitoring of the cell membrane is critically important. However, commercial fluorescence probes are stalked by the aggregation-caused quenching (ACQ) effect, and the reported aggregation-induced emission (AIE)-active probes are always limited by nonspecific aggregations in the biological environment. Herein, we report the rational molecular design of a state-of-the-art amphiphilic AIE luminogen (AIEgen), membrane tracker QMC12, using a core quinoline-malononitrile (QM) structure to suppress the ACQ effect, incorporate a positively charged pyridinium to regulate dispersity and strengthen the binding affinity to the negatively charged cell membrane, and extend the alky chain to improve the anchoring ability to the cell membrane. The membrane tracker QMC12, which disperses well in both hydrophilic and lipophilic environments, not only achieves minimal background interference and high signal-to-noise (S/N) ratio in the “ultrafast” visualization of the cell membrane, but also endows a “wash-free” characteristic. Furthermore, it realizes a spatial three dimensional (3D) view in a multicellular spheroid model and morphology changes over time. Moreover, QMC12 avoids false staining and signal loss and unprecedentedly achieves the direct observation of the cell membrane’s microstructure, which could elucidate spatiotemporal 3D model studies of the intercellular information exchange. Download figure Download PowerPoint Introduction The cell membrane, mainly composed of phospholipid bilayers, has a tremendous role in accurately coordinating various cellular behaviors.1–6 It is critically important to monitor the spatiotemporal changes of the cell membrane for early medical diagnosis and basic biological research.7–9 However, commercial fluorescence probes such as DiO or Dil are not ideal for cell membrane imaging because of their inherent aggregation-caused quenching (ACQ) effect, which often causes false signals and inevitable noises from an “always-on” pattern.10–13 Moreover, these commercial probes show poor solubility in both aqueous and lipid solutions, which complicates the staining procedures and limits further application at the multicellular level. In addition, their low positive charge density and weak hydrophobic interaction sometimes result in poor targetability and weak anchoring ability, thus providing inaccurate information feedback such as signal loss and unsustainable imaging (Figure 1a). Figure 1 | Rational design of the unique amphiphilic AIE-active probe for high-fidelity spatiotemporal mapping of the cell membrane. (a) Commercial probe DiO and Dil based on “always-on” pattern. (b) The rational design of the amphiphilic AIE-active probe to overcome the inherent deficiencies of commercial probes. The dimethylamino benzene group was used to extend wavelength, the pyridinium salt group for regulating solubility and targetability, and the long alkyl chain for promoting the anchoring property to cell membrane. (C) Left: schematic illustration of amphiphilic AIE-active QMC12 labelling cell membrane, thereby achieving “off–on” fluorescence upon enrichment in the cell membrane. Right: the interaction between the amphiphilic AIE-active probe QMC12 and a phospholipid molecule. Download figure Download PowerPoint Although design of fluorescence probes employing the concept of aggregation-induced emission (AIE) is highly desirable to overcome the “always on” pattern,14–24 most reported AIE luminogen (AIEgen)based probes still suffer from unexpected aggregations owing to their poor solubility in either aqueous or lipid environments, thereby providing inaccurate fluorescence signal. To resolve these issues, the water solubility of probes based on AIEgens must be improved to enable good dispersity in aqueous environment, decrease the background signal, simplify the operation procedure, and improve the multicellular level staining. Meanwhile, enhancement of the lipid solubility is expected to avoid false signals created by unexpected aggregation in lipid organelles. Furthermore, a positively charged group could be introduced, and the hydrophilicity and hydrophobicity can be adjusted to improve the affinity with the amphiphilic phospholipid bilayer to achieve targetability and anchoring ability to the cell membrane. In this work, we describe a rational design strategy to construct a novel amphiphilic AIE-active probe with a strong targeting ability and anchoring property for high-fidelity spatiotemporal imaging of the cell membrane. This amphiphilic AIEgen relies on the quinoline-malononitrile (QM) building block to overcome the ACQ effect, introduce the positively charged pyridinium salt to regulate the aggregation behavior in hydro- and lipophilic environments and generate strong binding affinity, and extend the alkyl chain to adjust the hydrophilicity and hydrophobicity (Figure 1b). With this strategy, the elaborated membrane tracker QMC12 posesses the following extraordinary features: (1) achieves high signal-to-noise (S/N) ratio with amphiphilic AIEgens through overcoming the ACQ effect and eliminating the undesired aggregations in hydro- and lipophilic environments; (ii) avoids false staining and signal loss with assistance of the superior targeting aggregation and excellent anchoring ability; (3) realizes ultrafast “wash-free” imaging because of superior water solubility with facilitated staining procedure; (4) spatiotemporally stains multicellular models because of the good dispersity and beneficial diffusion between cell membranes; and (5) maps morphology change over time with strong anchoring ability by tuning hydrophilicity and hydrophobicity to phospholipid bilayers (Figure 1c). In summary, the amphiphilic QMC12 has for the first time achieved 2D and 3D spatiotemporal visualization of the cell membrane without signal loss or false staining, even clearly observing the neurons dendrites’ microstructure, thus providing a promising alternative to commercial probes such as DiO or Dil for cell membrane imaging. Experimental Methods Materials and general methods All solvents and chemicals, unless specifically stated, were purchased commercially in analytical grade and used without further purification. 1H and 13C NMR spectra in deuterated solvent were obtained with a Bruker AvanceIII 400 MHz NMR spectrometer (Billerica, MA) using tetramethylsilane (TMS) as an internal standard. High-resolution mass spectrometry (HRMS) spectra were measured with a Waters LCT Premier XE spectrometer (Billerica, MA). Synthesis of compounds First, Py-QM was prepared by coupling the pyridine group with Br-QM through Suzuki reaction. Then, QM-PN and Py-QM-PN were synthesized by Knoevenagel condensation of QM and Py-QM with 4-dimethylaminobenzaldehyde. Finally, Py-QM-PN was treated with iodoethane and 1-iodododecane to obtain QMC2 and QMC12 after ion exchange. The detailed synthesis routes of all compounds were shown in Supporting Information Scheme S1. Cell lines The adenocarcinoma human alveolar basal epithelial cells (A549), human pancreatic cancer cell (PANC-1), adrenal pheochromocytoma cell (PC12), and human epithelioid cervical carcinoma cell (HeLa) were purchased from the Institute of Cell Biology (Shanghai, China). Cells were all propagated in T-75 flasks cultured at 37 °C under a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO/Invitrogen, Camarillo, CA), which was supplemented with 10% fetal bovine serum (FBS; Biological Industry, Kibbutz Beit Haemek, Israel) and 1% penicillin–streptomycin (10,000 U mL−1 penicillin and 10 mg mL−1 streptomycin; Solarbio Life Science, Beijing, China). In vitro cytotoxicity assay The cell cytotoxicity of QMC2 and QMC12 in HeLa cells was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were seeded into 96-well plates at a density of 1 × 104 cells/well and cultured at 37 °C under a humidified 5% CO2 atmosphere for 12 h. Then, the cells were exposed to various concentrations (1, 2.5, 5.0, 7.5, and 10 μM) of QMC2 and QMC12 or 100 μL culture medium as a negative control group. After incubation at 37 °C under a humidified 5% CO2 atmosphere for 24 h, MTT solution (5 mg/mL, 10 μL) was added to the media and incubated for another 4 h, and the absorbance at 490 nm was measured with a Multimode Plate Reader (BioTek, Burlington, VT). The relative cell viability (%) was calculated by the following formula: Cell viability (%) = mean absorbance value of the treatment group-blank/mean absorbance value of the control blank × 100. Cell imaging HeLa cells were seeded onto glass-bottom Petri dishes in culture medium (1.0 mL) and allowed to adhere for 12 h before imaging. Probe QM-PN, QMC2, and QMC12 at a final concentration of 5 × 10−6 M [containing 0.1% dimethyl sulfoxide (DMSO)] were added into culture medium and incubated for different time at 37 °C under a humidified 5% CO2 atmosphere. Cell imaging was captured by using a confocal laser scanning microscope (CLSM, Leica TCS SP8) with a 63× oil immersion objective lens. The fluorescence signals of cells incubated with probes were collected at 600–750 nm under excitation wavelength at 561 nm. Results and Discussion Incorporating pyridinium salt and long alkyl chain for amphiphilic AIE-active sensor To avoid the premature activated fluorescence caused by nonspecific aggregations in reported AIE sensors,25,26 we rationally designed and constructed an amphiphilic AIE-active probe step-by-step.27,28 First, the newly developed AIE building block, QM, was employed as the core structure to overcome the ACQ effect of commercial probes ( Supporting Information Figure S1), and the π-conjugated backbone dimethylamino benzene was utilized to extend the emission wavelength to provide QM-PN.29–33 Then, the electron-withdrawing pyridine group was introduced to further extend emission wavelength, affording the intermediate Py-QM-PN. As we expected, a significant fluorescence red shift of 111 nm was observed from QM to Py-QM-PN at solid state ( Supporting Information Figure S2). Sequentially, the ethyl group was covalently connected to the pyridine group, thus achieving positively charged QMC2, which was supposed to control the specific solubility in both lipophilic and hydrophilic systems with an initial “fluorescence-off” state and realize high targetability to the negativelycharged cell membrane through electron interaction.34–36 Finally, we extended the alkyl chain to tune the hydrophilicity and hydrophobicity, yielding membrane tracker QMC12, in which the anchoring ability could be strengthened by better hydrophobic interaction between the probe molecule and phospholipid bilayer of the cell membrane (Figure 1c). The molecular structures of all the designed compounds were confirmed by 1H NMR, 13C NMR, and HRMS. Amphiphilicity significantly minimizes undesirable fluorescence signal Previously developed AIEgen-based probes disperse well in either aqueous or lipid environments and thus are unable to achieve real targeting of the cell membrane; therefore, an amphiphilic AIE probe is expected to change this undesirable situation. To investigate the AIE and amphiphilicity properties of QM-PN and Py-QM-PN, their fluorescence spectra were first investigated in mixed water/tetrahydrofuran (THF) solvents with different fractions of water (fw). As shown in Figures 2a and 2b, the fluorescence intensity was enhanced when the water fraction gradually increased for both QM-PN (fw from 0% to 70%) and Py-QM-PN (fw from 0% to 60%), suggestive of their significant AIE property. The fluorescence intensity slightly decreased when the water fraction increased to 99%, which we attributed to the formation of nanoaggregates with a looser packing mode (Figure 2c). Both dynamic light scattering (DLS) (Figure 2d) and transmission electron microscopy (TEM) ( Supporting Information Figure S3) further supported that nanoaggregations formed in the aqueous environment (99% water). Obviously, QM-PN and Py-QM-PN inherit the excellent AIE features of the QM core, but they are largely hydrophobic and can form nonspecific aggregations in the aqueous system, thus still suffering from the “always-on” pattern for cell membrane imaging. Figure 2 | QMC12 exhibited “off–on” characteristic with amphiphilic behavior. (a–c) Fluorescence emission spectra and I/I0 plots of QM-PN (λex = 431 nm) and Py-QM-PN (λex = 442 nm) in a mixture of THF/water with different fw. I0 is the fluorescence intensity of QM-PN and Py-QM-PN in 0% water. (d) Size distribution of QM-PN and Py-QM-PN in a mixture of DMSO/Water (v/v = 1/99) obtained from DLS. Emission spectra of (e) QMC2 (10 μM) and (i) QMC12 (10 μM) in THF/water (λex = 447 nm). Emission spectra and I/I0 plots of (f and g) QMC2 and (j and k) QMC12 in Gly/water system with different Gly fractions (fg), λex = 454 nm. I0 is the fluorescence intensity of QMC2 and QMC12 in 99% water. Inset: fluorescence photographs of QMC2 and QMC12 in fg = 0 and fg = 99 taken under 365 nm UV irradiation. Size distribution of (h) QMC2 and (l) QMC12 (10 μM) in different solvents obtained from DLS. (m) HOMO and LUMO of QM-PN and QMC12 by DFT calculations. Download figure Download PowerPoint Compared with hydrophobic AIEgens (QM-PN and Py-QM-PN), QMC2 and QMC12, modified by a hydrophilic pyridinium salt unit, exhibited desirable amphiphilicity. Both showed extremely weak emission in all THF/water (Figures 2e and 2i), DMSO/water ( Supporting Information Figure S4), ethanol/water ( Supporting Information Figure S5) at any fraction of water, phosphate-buffered saline, and 10% FBS solution ( Supporting Information Figure S6). Those initial “fluorescence-off” states may result from their good dispersity in both aqueous and organic solvents, wherein QMC2 and QMC12 have free intramolecular motions and thus cause the fluorescence quenching according to the classic AIE mechanism. Moreover, DLS (Figures 2h and 2l) and TEM ( Supporting Information Figure S3) results show that QMC2 and QMC12 had extremely small sizes in both hydrophilic and lipophilic environments, proving their amphiphilic character. The AIE behaviors of QMC2 (Figures 2f and 2g) and QMC12 (Figures 2j and 2k) were further investigated in a glycerin (Gly)/water system through simulating the restriction of intramolecular motion (RIM) in high-viscosity environments. Both probes are non-emissive in aqueous solutions, but with increasing viscosity (fraction of Gly, fg = 0–99%), the fluorescence intensity gradually increased 31.2 times for QMC2 (Figure 2g) and 35.3 times for QMC12 (Figure 2k) via eliminating the non-radiative channel with the specific RIM mechanism. These results demonstrate that the unique amphiphilic QMC2 and QMC12 inherited the AIE property from the QM core and achieved the “fluorescence off” state in an aqueous system until it encountered the targeted site, which restricts the intramolecular motion, thus achieving high-fidelity imaging with a fluorescence “off–on” response. The wavelength extension from QM to QMC12 was realized by enhancing the electronic donor–acceptor effect, in which the absorption successfully shifted from 414 nm of QM to 455 nm of QMC12, and the emission peak red shifted from 514 nm of QM to 628 nm of QMC12 ( Supporting Information Figure S7).28 According to density functional theory (DFT) calculations the highest occupied molecular orbital (HOMO) electron density of QMC12 is mainly delocalized at the electron-rich N,N′-dimethylamino unit and phenyl group, and the lowest unoccupied molecular orbital (LUMO) is mainly delocalized at the pyridinium salt group (Figure 2m and Supporting Information Figure S8). The smaller HOMO–LUMO energy gap of QMC12 further confirmed the stronger electronic donating–accepting interaction, which is the reason for the longer absorption wavelength. It is highly expected that the amphiphilic probe QMC12 could overcome the ACQ effect from the “always-on” pattern as well as avoid undesirable aggregations in both hydrophilic and hydrophobic environments until encountering the high-viscosity cell membrane to emit fluorescence, thus it achieves “off–on” behavior in mapping the cell membrane. High-fidelity imaging of cell membrane Accurate imaging of the cell membrane has great importance for tracing cell distribution and reflecting the structural integrity.37 Considering the positively charged sensor could accumulate at the negatively charged cell membrane and the long alkyl chain could anchor to the phospholipid bilayers, the QMC12 is expected to provide high-fidelity imaging of cell membrane. Here, all the designed molecules were evaluated by co-staining with commercial membrane tracker DiO in HeLa cells (human epithelioid cervical carcinoma cells). As shown in Figures 3a and 3b, the lipophilic probes QM-PN and Py-QM-PN showed obvious fluorescence signal in the cytoplasm rather than the cell membrane, resulting in poor Pearson’s correlation coefficient of 0.04 for QM-PN and 0.12 for Py-QM-PN ( Supporting Information Figure S9). In addition, the intensity profile of the linear region of interest (ROI) across the cells showed that QM-PN and Py-QM-PN do not overlap well with the commercial DiO, further demonstrating the poor co-localization ability (Figures 3a and 3b). These results indicate that the lipophilicity of QM-PN and Py-QM-PN make them more favorably penetrate through the cell membrane and aggregate as AIE nanoparticles to emit fluorescence in the cytoplasm. In contrast, the positively charged QMC2 and QMC12 displayed bright fluorescence signal in the cell membrane rather than the cytoplasm or extracellular matrix, which confirmed the targeting ability of the positively charged sensor towards the negatively charged cell membrane (Figures 3c and 3d). It is noted that the QMC2 with short alkyl chain exhibited a smaller Pearson’s correlation coefficient of 0.45 (Figure 3c and Supporting Information Figure S9) than the correlation coefficient of QMC12 (0.88), suggesting a stronger anchoring property with longer alkyl chain (Figure 3d and Supporting Information Figure S9). In addition, several cell lines’ cell membranes were stained well using QMC12 probe with wide application range ( Supporting Information Figure S10). These results strongly support the remarkable cell membrane targeting ability of amphiphilic QMC12. Figure 3 | High-fidelity imaging of cell membrane by QMC12. (a–d) HeLa cells were incubated with QM-PN (5 μM), Py-QM-PN (5 μM), QMC2 (5 μM), and QMC12 (5 μM) for 20 min followed by co-staining with DiO (10 μM) for 20 min. (a2–d2) Green channels from DiO (λex = 488 nm, λem = 500–600 nm). (a3–d3) Red channels from QM-PN, Py-QM-PN, QMC2, or QMC12 (λex = 561 nm, λem = 600–750 nm). (a4–d4) Merged images of green, red, and bright field channels. (a5–d5) The intensity profile of the white linear ROI across the cell in a4–d4. All images share the same scale bar of 10 μm. Download figure Download PowerPoint To further confirm that the amphiphilicity of QMC12 can eliminate undesirable aggregations and the proper hydrophilicity and hydrophobicity can ensure strong anchoring ability to phospholipid bilayers, we compared the cell membrane imaging performance of QMC12 with commercial trackers. Due to excessive lipid solubility and poor anchoring ability, both commercial DiO and Dil sensors showed undesired false staining (Figure 4a and Supporting Information Figure S11) and signal loss (Figure 4b). In contrast, amphiphilic QMC12 would not accumulate on bubbles with less electrostatic interaction, thus successfully avoiding a false signal (Figure 4a). Moreover, due to the effective electrostatic interaction and the strong anchoring effect of the long alkyl chain between cell membrane and probe, QMC12 could avoid signal loss after several experimental operations (Figure 4b). These results indicate that QMC12 has realized high-fidelity imaging of cell membrane with high S/N ratio. Figure 4 | High-fidelity 3D imaging of cell membrane. (a) False signal and (b) signal loss of DiO compared with QMC12. All images share the same scale bar of 10 μm. (c) Z-stack images of Hela cells from 0–9 μm after staining with QMC12 (5 μM) for 20 min. All images share the same scale bar of 10 μm. (d) The 3D reconstructed images of Hela cell. (e) Z-stack images of PC12 cells from 0 to 9 μm after staining with QMC12 (5 μM) for 20 min, followed by staining with Hoechst 33342 (20 mM). The overlay channel is merged bright field, blue channel (λex = 405 nm, λem = 420–450 nm), and red channel. All images share the same scale bar of 10 μm. (f) The 3D reconstructed images of PC12 cell. Download figure Download PowerPoint Encouraged by the above cell imaging experiments, more detailed three-dimensional (3D) structural information of the cell membrane was expected to be obtained. Therefore, we recorded a series of confocal images (at different depth) by scanning the HeLa cells (Figures 4c and 4d) and PC12 cells (a common nerve cell line, Figures 4e and 4f) stained with QMC12. As expected, the red fluorescence signal of QMC12 overlapped well with the cell membrane observed in the brightfield images of HeLa cells (Figure 4c) and PC12 cells (Figure 4e), thus showing the 3D morphology as a protective barrier surrounding cells. Surprisingly, the cell membrane was continuous and uninterruptedly stained, even to the extent that the dendrites’ microstructure in neuron cell was clearly observed (Figure 4f and Supporting Information Video S1), which characteristic is helpful to observe the interaction between cells, especially the direct information exchange between nerve cells. Taken together, compared to commercial cell membrane trackers, our designed amphiphilic AIE-active probe QMC12 realized high-fidelity 2D and 3D cell membrane imaging with a much higher S/N ratio. Ultra-fast cell membrane staining with “wash-free” behavior The fragility of the cell membrane urgently requires uncomplicated staining procedures. However, the commercial DiO probe (work concentration of 10−3 M) always needs several dissolution and washing procedures because of its poor solubility in both aqueous and organic solvents ( Supporting Information Figure S12).38 The amphiphilic AIEgen QMC12 should disperse well in both aqueous and lipid solutions with an initial “fluorescence-off” property, so as to generate potential “wash-free” behavior, thereby facilitating experimental efficiency and avoiding the signal-loss caused by the postwashing procedure. As shown in Figure 5, obvious fluorescence signal was observed outside the cel