Visualization of supramolecular assembly by aggregation‐induced emission

Supramolecular architectures are constructed by the self‐assembly of small building blocks via the use of metal‐ligand coordination, π–π stacking interactions, hydrogen bonding, host‐guest interactions, and other noncovalent driving forces, which confer unique dynamic reversibility and stimulus responsiveness to the supramolecular materials and also lead to the demand of expensive and complex equipment for the characterization of supramolecular assembly processes. Fortunately, the self‐assembly processes bring the monomeric chromophores together, offering possibilities to establish ties between the supramolecular assembly and aggregation‐induced emission (AIE) techniques. Compared to conventional luminescent molecules, AIE luminogens (AIEgens) exhibit significant fluorescence enhancement upon the restriction of molecular motions, thus displaying the advantages of signal amplification and low background noises. Given the above, the real‐time, sensitive, and in situ visualization of the formation of self‐assemblies and their stimuli responsiveness based on AIE becomes accessible. Here, we review recent works that encompass the visualization of supramolecular assembly‐related behaviors by means of AIE characteristics of chromophores. The organization of this review will be by different types of supramolecular architectures, including metallacycles/cages, micelles/vesicles, supramolecular polymers, and supramolecular gels. An overview of future opportunities and challenges for the real‐time monitoring of supramolecular assembly by AIE is also provided.


INTRODUCTION
Self-assembly is capable of driving the molecular building blocks to form well-defined supramolecular architectures by noncovalent interactions, such as electrostatic attractions, metal-ligand coordination, π-π interactions, hydrogen bonding, and host-guest interactions. [1,2] During the past decades, the supramolecular assembly has witnessed significant development in many fields after it was first proposed. [3][4][5] Up to now, supramolecular topological structures with different dimensions and sizes have been obtained, from the nanoscale to microscale, from one-dimensional fibers to twodimensional sheets to three-dimensional frameworks. These supramolecular architectures have been widely used for drug vehicles, electronic devices, medical diagnosis, catalysis, and imaging. [6][7][8][9][10] Companying with the development of supramolecular chemistry, thorough and precise analysis of supramolecular assembly has also been a burning topic. Numerous methods have been developed by utilizing transmission electron microscopy (TEM), [11][12][13] scanning electron microscope (SEM), [14,15] isothermal titration calorimetry (ITC), [16,17] nuclear magnetic resonance (NMR), [17][18][19] ultraviolet-visible spectroscopy (UV-Vis), [20,21] infrared spectroscopy (IR), [22,23] circular dichroism (CD), [24,25] and atomic force microscope (AFM) techniques, [26] etc., to understand the structure, morphology, and formation mechanism of supramolecular assemblies. For example, Guilleme et al. combined CD, SEM, TEM, and computational studies to reveal the formation of non-centrosymmetric homochiral columnar subphthalocyanine assemblies. [27] These achievements represent important milestones that promote the development of supramolecular chemistry. However, the dynamic nature of supramolecular assembly and cumbersome sample preparation for self-assembly analysis under these precise instruments cause difficulties in monitoring the supramolecular assembly-related behaviors in situ. In this regard, the characterization of supramolecular assemblies and monitoring their corresponding dynamic processes remain appealing yet significantly challenging.
The fluorescence detection strategy is promising for the visualization of self-assembly processes, which can provide pronounced emission signals for reflecting the changes in molecular conformations, intermolecular forces, and the environment of the fluorophores. It has been employed in chemistry, cell biology, and biomedical fields with excellent in situ space, real-time, high sensitivity, and selectivity properties. [28][29][30][31] The supramolecular assembly process can involve aggregates of chromophores. Unfortunately, conventional fluorescent molecules often suffer from aggregationcaused quenching (ACQ) effect upon self-assembly, which leads to weakened emission and reduced signal sensitivity for their practical applications. [32] In 2001, the Tang group reported an aggregation-induced emission luminogen (AIEgen) that exhibited weak fluorescence in solution but showed bright emission in the aggregate state. On this basis, the concept of aggregation-induced emission (AIE) was established and has been widely explored in areas from bioimaging, optical sensing, anti-counterfeiting to optoelectronic devices. [33][34][35][36] In a broader context, the foundation of this field has connections to some earlier contributions that help enrich AIE research. [37] For example, in 1936, Scheibe [38] and Jelley [39] independently found that the aqueous solutions of pseudoisocyanine can exhibit a new emission band with increased fluorescence intensity upon increasing the concentration, which was attributed to the formation of J-aggregates. [40][41][42] Another example is the intercalation of dyes into nucleic acids that leads to significantly enhanced dye fluorescence due to the rigidification of the molecules. [43][44][45][46][47] These intriguing phenomena have attracted more attention after the concept of AIE was proposed and intensively exploited. AIEgens are useful for real-time visualization since it solves the ACQ problem and endows fluorophores with bright emissions once the condensed states are formed. [48,49] AIEgen has been used to directly visualize the interfacial dynamic self-assembly, [50] differentiate the microphase separation in immiscible polymer blends, [51] measure the solubilization locations in the micelles, [52] observe the enantiorecognition and resolution, [53] and monitor the responses to external stimuli. [54] Ascribed to their intrinsic advantages, including bright emission in aggregates, high photobleaching threshold, and high signal-to-noise ratio, AIEgens have attracted extensive attention and provide new opportunities for visualizing supramolecular self-assembly. [35,55] Given the above, the development of monitoring supramolecular assembly-related processes through AIE has been started. Many contributions have been made, and some of them are highlighted in reviews. [56][57][58][59][60][61] For example, Tang and co-workers summarized the basic concepts and advances in the construction and applications of AIEgenbased supramolecular materials, which involves researches on visualization of the supramolecular assembly process. [62] Yang et al. reviewed different AIE-active supramolecular assemblies for sensing and detecting different analytes, which encompass studies associated with AIEgen-based visualization. [63] Nevertheless, it is still lacking a comprehensive review to summarize the recent progress in this emerging area. Herein, we provide coverage of the recent developments of AIEgens applied in the visualization of dynamic processes associated with the supramolecular assembly. The organization of this review will be by different selfassemblies, including metallacycles/cages, micelles/vesicles, supramolecular polymers, and supramolecular gels. It aims to provide insight into the mechanism of self-assembly based on AIE techniques in chemistry, physics, biology, and biomedical applications (Figure 1). An overview of future opportunities and challenges associated with the visualization of supramolecular assembly by means of AIE is also provided.

Visualization of the metallacycles
Metallacycles are discrete and well-defined two-dimensional metal-organic complexes driven by coordination interactions between ligands and metal ions (silver, gold, cadmium, platinum, etc.). [64][65][66][67][68][69] The molecular motions of ligands can be effectively restricted by constructing metallacycles, thereby activating the AIE properties. As a result, the formation of metallacycles or the self-assembly processes related to them is probably able to be monitored by exploiting the AIE signal changes.
F I G U R E 1 Visualization of supramolecular assembly by AIE. Reproduced with permission. [79] Copyright 2020, Wiley-VCH GmbH. Reproduced with permission. [87] Copyright 2020, Wiley-VCH GmbH. Reproduced with permission. [106] Copyright 2019, The American Chemical Society. Reproduced with permission. [109] Copyright 2022, Wiley-VCH GmbH. Reproduced with permission. [122] Copyright 2020, Wiley-VCH GmbH In 2017, Hahn et al. reported dinuclear silver(I) and gold(I) metallacycles (SA1), which were prepared from the coordination-driven self-assembly of tetraphenylethylene (TPE) bridged N-Heterocyclic carbenes (M1) and metal ions, viz. Ag(I) and Au(I) (Figure 2A). [64] In dilute solutions, M1 showed weak emissions with fluorescence quantum yield (Φ F ) lower than 1%. However, SA1 could exhibit bright fluorescence with Φ F reaching up to 47%. Such a sharp contrast originated from the restriction of phenyl group rotations in M1 by metal coordination. Interestingly, it was demonstrated that there was no participation of Ag(I) or Au(I) in the SA1 excited states. As a result, the photophysical properties of SA1 were independent of the metal ions. Compared to the ligand precursor, AIE metallacycles in this work had distinct photoluminescence (PL) enhancement in dilute solutions due to metalation, thereby leading to real-time monitoring of the metallacycle formation. Later on, the Han group designed two similar AIE metallacycles (SA2, Figure 2B) based on M1 derivatives with benzimidazole units and Ag(I)/Au(I) ions. [70] As expected, SA2 gave high Φ F values of up to 55% in dilute solutions due to the rigidification of the TPE skeleton upon metalation. Furthermore, SA2 could display enhanced fluorescence signals when water was added into their acetonitrile solutions with fractions increased from 0 to 60%. It is reasonable since the water, as a poor solvent, could cause aggregation of SA2 for further inhibition of TPE intramolecular motions. However, as the water fraction increased to >70%, the PL intensity of SA2 began to decrease and blueshift, presumably owing to the intramolecular charge transfer process in SA2. In particular, SA2 carrying coumarin units could undergo [2 + 2] cycloaddition under highpressure mercury lamp irradiation (λ = 365 nm) at ambient temperature for 20 h to form new metallacycles, which had high Φ F values (23%) compared to their ligands (Φ F < 1%).
Recently, Li and co-workers designed and synthesized a new family of TPE-cored ligands (M3-1, M3-2, and M3-3) containing a conjugation of TPE with terpyridine units to chelate with Cd(II) ions for the fabrication of rosettelike metallacycles ( Figure 2C). [67] M3-1 with two terpyridine substituents binding with Cd 2+ ions could form a mixture of metallacycles (SA3-1). This result was attributed to the flexible angle between two terpyridine units in M3-1 that led to different metallacycles. However, SA3-1 showed weak fluorescence since their peripheries still had free-rotating phenyl groups. M3-2 and M3-3 exhibited stronger emissions owing to their additional bulky terpyridine units, which partially restricted the intramolecular motions of TPE. Moreover, M3-2 and M3-3 could coordinate with Cd 2+ ions and thereby form discrete macrocycles, viz., a double-layered hexameric rosette (SA3-2) and a triple-layered heptameric rosette (SA3-3), respectively. As a result, the TPE units in SA3-2 and SA3-3 were further immobilized, leading to severalfold enhanced Φ F values of the macrocycles compared to their ligands. Such turn-on fluorescence changes provided a new approach for monitoring the formation of SA3-2 and SA3-3. In another work, the same group developed another highly emissive metallacycle, which was formed from the coordination of Zn 2+ ions with a TPE-based derivative bearing terpyridine groups. The differences were that the ligand suffered from ACQ effects, but the metallacycle could realize the AIE phenomenon in acetonitrile/methanol (MeOH) mixtures. The above examples illustrated a series of metallacycles self-assembled from different metals, that is, Ag(I), Au(I), Cd(II), Zn(II), and various TPE derivatives. By the mean of AIE characteristics, they displayed visible fluorescent changes compared to their starting materials, resulting in the monitoring of the formation of these metallacycles.
By further self-assembling AIEgen-based metallacycles and other components, the fluorescence properties will also change, which is possible to detect the presence of new analytes visually. Yang and his colleagues reported a fluorescent hierarchical assembly formed by multiple electrostatic F I G U R E 2 (A) Chemical structures of M1 and SA1. Reproduced with permission. [64] Copyright 2017, Wiley-VCH GmbH. (B) Chemical structure and photographs of SA2 at different water content. Reproduced with permission. [70] Copyright 2019, Wiley-VCH GmbH. (C) Chemical structures of M3-1, M3-2, and M3-3, as well as schematic illustration of the self-assembled metallacycles of SA3-1, SA3-2, and SA3-2. Insets: photographs of SA3-2 and SA3-2 in CH 3 CN/H 2 O. Reproduced with permission. [67] Copyright 2018, Springer Nature F I G U R E 3 (A) Chemical structures of M4-1 and M4-2, as well as schematic representation of the possible binding and aggregation mode of SA4 and heparin. Insets: photographs of heparin (left), SA4 (middle), and their mixture (right). Reproduced with permission. [71] Copyright 2015, The American Chemical Society. (B) Chemical structure of SA5, as well as schematic representation of the formation of 3D biohybrid complexes via the self-assembly of SA5 and TMV. Insets: photographs of SA5 before and after adding TMV. Reproduced with permission. [73] Copyright 2016, The American Chemical Society. (C) Chemical structure of SA6. (D) TEM images of NP1, NP2, and VS, as well as CLSM images of the HeLa cells incubated with NP1, NP2, and VC for 8 h. (E) H&E and Ki67 analyses of tumor tissues after various formulations. Reproduced with permission. [74] Copyright 2017, The American Chemical Society interactions of positively charged organoplatinum(II) metallacycles (SA4-1) and negatively charged heparin ( Figure 3A). [71] By chelating 120 • TPE-containing donor (M4-1) with 120 • diplatinum (II) acceptor (M4-2), a hexagonal metallacycle SA4 bearing three TPE units was first synthesized ( Figure 4A). It possessed AIE characteristics that were capable of monitoring the supramolecular assembly-related behaviors process. Upon addition of the heparin to acetone and water mixtures of SA4, remarkable PL enhancement at the wavelength of 486 nm was observed since the molecular motions of TPE units were restricted by self-assembly. When the concentration of heparin increased to a high level, SA4 could further organize to produce entangled 3D networks. It was proposed that a series of SA4 monomers were anchored on heparin chains, which resulted in complexes that could intertwine mutually via the remaining electrostatic interaction sites. Based on the dramatic fluorescence changes in the presence or absence of heparin, SA4 was used as a turn-on probe for the quantification of heparin. The detection range of 0-28 μM covered the clinical dosage level.
In recent years, the Stang group created a series of AIE-active platinum(II) metallacycles with increasing complexity and functionality for biosensing, bioimaging, drug Reproduced with permission. [77] Copyright 2015, Springer Nature. Reproduced with permission. [78] Copyright 2020, Wiley-VCH GmbH. (B) Chemical structures and assembly process of M9, SA9-1, SA9-2, and SA9-1; (C) Photographs of SA9-2 in CH 3 CN/H 2 O mixtures with different water content and SA9-2 solution taken during the reaction at different times. Reproduced with permission. [79] Copyright 2020, Wiley-VCH GmbH. (D) Chemical structure and schematic illustration of M10-1 and SA10-1. (E) Chemical structure and schematic illustration of M10-2 and SA10-2. Reproduced with permission. [80] Copyright 2019, the American Chemical Society delivery, white-light-emitting, and so on. [72][73][74][75] AIE units in these metallacycles exhibited bright emissions when further self-assembly occurred. In 2016, by multiple electrostatic interactions, Stang and co-workers employed a TPE-based platinum(II) metallacycle SA5 to interact with 1D rod-like tobacco mosaic virus (TMV) to prepare 3D biohybrid complexes ( Figure 3B). [73] When negatively charged TMV was added to the water/dimethyl sulfoxide solution of the positively charged SA5, its hydrodynamic radius (R h ) increased greatly from 18 to 618 nm, indicating the formation of large aggregates. Meanwhile, remarkable fluorescence enhance-ment at 490 nm was observed due to the nanoconfinement effect on TPE in the biohybrid complexes. Such fluorescence changes allowed the self-assembly process of TMV and SA5 to be visualized in situ. Subsequently, Stang and coworkers constructed an amphiphilic polymer SA6 with AIE characteristics. [74] It consisted of a TPE-based platinum(II) metallacycle as the core and glutathione (GSH)-responsive diblock copolymers as the arms ( Figure 3C). Using the amphiphilicity and AIE effect, SA6 could self-assemble into highly emissive nanoparticles (NPs) with different sizes (NP1 and NP2) and vesicles (VC) via different techniques for live cell imaging ( Figure 3D). The assemblies displayed various endocytic pathways, different internalization rates, and disparate cytotoxicities toward HeLa cells owing to their diverse morphologies and sizes. Furthermore, the NPs and VC were able to encapsulate anticancer drugs neutral doxorubicin (DOX) and doxorubicin hydrochloride, respectively, which could be released by the GSH-triggered decomposition of the arms on SA6. As a result, the DOXloaded nanoparticles could inhibit the growth of tumors and achieve imaging-guided synergistic drug therapy for cancer ( Figure 3E).

Visualization of the metallacages
Metallacages are also built by the coordination-driven selfassembly of metal ions and ligands, representing another discrete and well-defined metal-organic complexes and possessing the structures of three-dimensional tetrahedra, dodecahedra, or other polyhedra. [65,76] The formation of metallacages could be studied by introducing AIEgens into the monomers as fluorescence indicators.
In 2015, Stang and co-workers employed a metal acceptor cis-Pt(PEt 3 ) 2 (OTf) 2 (M7-4), a TPE-cored ligand (M7-1), and benzene dicarboxylates (M7-2 and M7-3) to prepare tetragonal platinum(II) metallacages SA7-1 and SA7-2 ( Figure 4A). [77] M7-1 had no emission in the dichloromethane (CH 2 Cl 2 ) solution owing to the energy dissipation by intramolecular rotations of TPE. However, after locking M7-1 into the rigid metallacages, bright yellow fluorescence appeared in the CH 2 Cl 2 solution with Φ F reaching up to 23.2% for SA7-1 and 10.8% for SA7-2, respectively. Moreover, with the increase of the relative content of hexane in the solution, SA7-1 gradually aggregated, and the Φ F value continuously increased to 60.6%, accompanied by emission color changes from yellow to blue to cyan and morphological changes from irregular nanoparticles to regular nanospheres to net-like aggregates. In contrast, SA7-2 had less pronounced fluorescence changes and morphological changes than SA7-1, which was attributed to the dispersive effect of poly(ethylene glycol) chains on M7-3. The observations indicated that SA7-1 and SA7-2 were still AIE-active, thereby releasing intense fluorescence on aggregation. On the one hand, the formation of aggregates further restricted the conformational changes of M7-3. On the other hand, solvents with low polarity could inhibit the metal-to-ligand charge transfer process. Apparently, the emission color changes of metallacages during aggregation can be used to estimate the aggregation degrees.
Recently, the Yan group tracked an efficient metallacage to metallacage transformation process by monitoring the fluorescence signal changes. A two-component metallacage SA8-1 was prepared by the coordination-driven self-assembly of M7-1 and M7-4. [78] Subsequently, another two-component metallacycle (SA8-2) was added to the SA8-1 solution, which resulted in a supramolecular fusion that produced a three-component metallacage SA8-3 ( Figure 4A). The entire conversion process was easy to monitor by measuring the fluorescence signals of the supramolecular system in real-time. SA8-1 exhibited strong fluorescence via the suppression of M7-1 intramolecular motions upon coordination. However, the formation of SA8-3 could partially quench the fluorescence and redshift the emission maximum from 490 to 505 nm due to the heteroligation of the platinum center. According to the theoretical calculation, photoinduced electron transfer between TPE and the 4,4-bipyridine motif contributed to the fluorescence signal changes during the cage-to-cage transformation process, because the lowest unoccupied molecular orbitals of SA8-3 located on the bipyridine rings.
Based on the previous report on metallacycles (SA1), Han and Hahn et al. further prepared several discrete Ag metallacages through coordination-driven self-assembly of M1 derivatives (M9). [79] The N-substituents greatly influenced the mode of the self-assembly, which led to different metallacages, including SA9-1 and SA9-2 ( Figure 4B). Their structures were confirmed by single crystal X-ray diffraction analyses. The metallacages showed enhanced fluorescence due to the restriction of the TPE motifs upon coordination ( Figure 4C). Under UV irradiation in the air, oxidative photocyclizations of SA9-1 and SA9-2 could occur at the tetrakisarylethylene unit to yield SA9-3 complexes. Note that all the products, that is, SA9-3 with different alkyl chains shared the same tetranuclear metallacage structure although the overall structures of the precursors (SA9-1 and SA9-2) were quite different from each other. SA9-3 involves the 9,10-phenyl-substituted phenanthrene bridge, which was ACQ-active and responsible for immobilizing the tetrakistriazolylidene ligands, thereby resulting in the fluorescence intensity decreases ( Figure 4C). The M9-metal coordination and photoinduced structural transformation induced distinct luminescence changes in the supramolecular systems, allowing us to monitor the self-assembly evolution conveniently.
The above examples described the visual monitoring of the formation of metallacages by locking AIEgens in their rigid skeleton. In addition to this approach, another strategy inspired by green fluorescent protein was designed by anchoring AIEgens within the confined cavities of metallacages to understand their formation. In 2019, the Stang group prepared two different metallacages, SA10-1 and SA10-2, with exoand endo-functionalized TPE moieties ( Figure 4D,E). [80] As expected, SA10-1 and SA10-2 exhibited enhanced fluorescence compared to their corresponding ligands because of the increased local concentration of TPE units. However, in dilute solution, SA10-2 as the endohedral functionalized metallacage emitted much stronger fluorescence than that of SA10-1 since the former enclosed 24 TPE units within the cavity that could restrict TPE more severely than the outer surface. Therefore, the luminescence from the confined cavity could be more sensitive to monitor the formation of endohedral functionalized metallacages.
In addition to visualizing the formation process of metallacages, further self-assembly of AIE-based metallacages could be used for observing the material responses to external stimuli (such as chemical or biological species, temperature, pH, light, and so on) with the fluorescence changes of AIEgens. The Stang, Chen, and Huang group reported a highly emissive TPE-based metallacage SA11, which was prepared from multicomponent coordination-driven selfassembly of M7-1, M11-1, and M7-4 ( Figure 5A). [81] Two variants of the 2-distearoyl-phosphatidylethanolamine (DSPE)/polyethylene glycol (PEG) conjugate, mPEG-DSPE, and biotin-PEG-DSPE, were used as surfactants to further self-assembly with the metallacages for improving their  [82] Copyright 2020, the American Chemical Society. (E) Chemical structures of M12-1, M13-1, and M7-4, as well as schematic illustration showing the formation of SA13 and self-destruction of SA13 with amino acid. (F) Fluorescence spectra of SA13 with different concentrations of glutathione and cysteine, as well as plots of concentrations and fluorescence intensities at 500 nm. Reproduced with permission. [83] Copyright 2017, the American Chemical Society circulation time and accumulation in tumors for cancer treatment. The vitro studies demonstrated that the SA11contained nanoparticles (MNPs) had excellent targeting ability and could be selectively delivered to cancer cells that overexpressed the biotin receptors through receptormediated endocytosis. In vivo, the highly emissive MNPs displayed intense fluorescence signals in tumor tissues, which realized visual tracking of the tumor location ( Figure 5B). Particularly, MNPs also had higher antitumor efficacy and lower toxicity than free platinum anticancer drugs, that is, oxaliplatin, carboplatin, and cisplatin. These results suggest the potential of AIEgen-based metallacages for the next generation of nanomedicines.
Later on, The Zhang group reported two metallacages that were prepared from a TPE benzoate (M12-1) and two different bipyridine columns, M12-3 and M12-4, of which M12-4 carried two rhodamine B units ( Figure 5C). [82] There was a good spectral overlap between the absorption band of M12-1 and the emission band of ligand M12-3, resulting in Förster resonance energy transfer (FRET) behaviors from M12-3 to M12-1 in SA12-1. However, M12-1 served as an excitation energy donor in SA12-2, involving a reverse FRET from M12-1 to M12-4. Hence, the two metallacages possessed different photophysical properties. By changing solvents, mechanical forces, and temperature, the metallacages could exhibit very different emissions, which were attributed to the changed molecular packing and donor/acceptor photophysical behaviors in different external conditions. As a result, the metallacages with external stimuli responsiveness could not only distinguish alcohols with similar structures, but also be used as fluorescent proportional thermometers ( Figure 5D).
If the fluorescence properties of metallacages are sensitive to solvent solubility and polarity, they can be employed to construct sensors or probes for visualizing microenvironment changes of supramolecular assemblies. Stang et al. reported a sensor with AIE characteristics for detecting amino acids by a self-destructive mechanism. [83] An emissive tetragonal prismatic metallacage (SA13) was prepared by chelating 90 • Pt(II) acceptors (M7-4) with bipyridyl ligands (M13-1) and sodium benzoate of TPE (M12-1). It was nearly nonemissive in MeOH/H 2 O (1/1, v/v), but the fluorescence was significantly enhanced when amino acids were added. The Pt─N coordination bonds of SA13 can be disrupted by adding thiol-containing amino acids since the formed Pt─S bonds are stronger than the relatively dynamic Pt─N bonds. Such a reaction could release the TPE-based benzoate acid, which had poor solubility in MeOH/H 2 O (1/1, v/v), thereby activating AIE. Detailly, upon adding glutathione and cysteine, the emission intensities of SA13 increased linearly with the amino acid in the concentration range of 0-80 μM, corresponding to their detection limits of 1.87 × 10 −7 and 2.78 × 10 −7 M (S/N = 3), respectively.

VISUALIZATION OF THE MICELLES AND VESICLES
In the aqueous solution, the monomeric amphiphilic molecules form micelles or vesicles by hydrophobic interactions once the concentration of monomers reaches the critical aggregation concentration (CAC). [84,85] The micelles or vesicles could present different self-assembly structures, including spherical shapes, ellipsoidal shapes, rod-like shapes, and extended sheet structures. By introducing the AIEgen-based amphiphiles, the visualization of micelle or vesicle formation and their morphology transitions were realized. Additionally, the applications of micelles and vesicles, that is, drug delivery, also can be investigated by tracking the AIE signal changes during the variation of self-assembly states. [86] In 2019, Tang et al. successfully synthesized and separated stereogenic amphiphiles Z-M14 and E-M14 with TPE core and oligoethylene glycol monomethyl ether (OEG) tails ( Figure 6A). [55] Z-M14 could self-assemble into vesicles (Z-SA14-1), while its E-counterpart formed micelles (E-SA14-1) in water. Based on the AIE features, the initial step (i.e., CAC) of their concentration-dependent self-assembly could be detected, which is much lower than that measured by the conventional transmittance measurement. Moreover, the (Z)-amphiphile had a very sensitive thermoresponsive behavior, which involved dissolution/vesiculation of the isomers and dehydration/hydration of the OEG chains ( Figure 6B). Therefore, its phase transition under different temperature ranges could be visualized under confocal laser scanning microscopy (CLSM) via AIE. The result showed that as the temperature rises from 37 • C to 41 • C, the fluorescence intensity of the vesicles decreases due to the dissolution of the (Z)-amphiphile. However, bright spots appeared gradually with the temperature ascending from 41 • C to 45 • C because of dehydration of the OEG tails that resulted in aggregation of Z-M14. It is believed that this work for visualization of the initial step of self-assembly and phase transition by AIE will pave the way for more clearly understanding the dynamic processes of supramolecular assembly.
In 2020, Li and co-workers reported a new strategy for realtime monitoring nanoparticle dynamics. [87] They combined AIE and ACQ effects by modifying the corresponding fluorophores on an amphiphilic dendrimer (M15), which could self-assemble into micelles (SA15) in water ( Figure 6C). Therefore, two entirely different fluorescence colors would be alternatively activated during the micellar transformation. According to the vitro experiments, the fluorescence intensity ratio of I AIE /I ACQ was dramatically changed at different states of the micelles, of which the intact state had higher I AIE /I ACQ values and the dissociated state resulted in lower values. On this basis, fluorescence ratio maps could be constructed via CLSM by recording continuous fluorescence images of the micelles in living cells or different tissues, which allowed quantitative analysis of the state distribution of M15 in the complex physiological environment ( Figure 6D). Benefiting from this strategy, the understanding of delivery mechanisms of the micellar nanostructures would be deepened, thereby providing the theoretical framework for biochemical research of supramolecular assemblies.
Tang and Qin et al. designed a cationic amphiphilic probe (M16) with AIE properties and excited-state intramolecular proton transfer processes for detecting anionic surfactants with high sensitivity (Table 1). [88] When M16 interacted with the anionic sodium dodecylbenzenesulfonate (SDBS) via electrostatic attractions and hydrophobic interactions, micelles (SA16) formed at a very low concentration of SDBS (2.39 μM). This activated AIE signals of M16, thereby visualizing the micellization and probing the existence of SDBS. On the other hand, M16 emitted green fluorescence (510 nm) in "keto" state, but had negligible "enol" fluorescence signals (450 nm) in aqueous solution. The catanionic aggregates kept the intramolecular hydrogen bond of M16 from disruption, resulting in a higher keto/enol emission ratio and thus improving the sensitivity of the AIE probe. By employing the signal-to-noise ratio approach, a detection limit for SDBS was evaluated to be 0.051 μM, which is much lower than the detection limit of traditional methods. Unlike the aforementioned examples, this work utilized an AIE amphiphilic building block to detect common anionic surfactants, offering a universal and efficient strategy for surfactant detection in environmental monitoring.
In 2015, Tang and Lu et al. inserted the TPE core into sodium dodecyl sulfonate to create a surfactant (M17) with AIE characteristics for achieving the visualization of the transition processes of micelles (Table 1). [89] The change of aggregation state from M17 to micelles (SA17) could be monitored by a plot of its fluorescence intensity maximum at 490 nm versus the concentration of M17 with an inflection point at ca. 30 μM, which is consistent with the result measured by the conductivity variation method. Under the confocal fluorescence microscopy (CFM), SA17 micelles were seen as luminescent dots by the mean of diffraction blur. Since neutral electrolytes can affect the electrical double layer of the micelles, the morphology changes of SA17 from dot, to rod, to wormlike structures occurred when NaCl was added. Importantly, the structural transition processes could TA B L E 1 Summary of the partial AIE-based micelles and vesicles for visualization  Reproduced with permission. [87] Copyright 2020, Wiley-VCH GmbH be directly observed by CFM, which was further evidenced by other techniques, including dynamic light scattering and TEM. This work also involves the measurement of the critical distance between TPE cores for activating AIE, providing support to the widely accepted mechanism of AIE, viz. restriction of intramolecular rotation. In another work, Lu and co-workers used M17 to visualize the interaction between surfactants and polymers by CFM (Table 1). [90] Compared with the conventional methods, that is, viscosity and conductivity measurement, the fluorescence intensity changes of the self-assemblies in CFM gave an intuitive insight into the CAC and polymer saturation point (PSP). To be specific, M17 attached to the chitosan formed micelles (SA18) above the CAC, and simultaneously the polymer chains shrank, which restricted the intramolecular motions of M17. In contrast, above the PSP, the intensity changes tended to level off after reaching PSP since chitosan chains were saturated with M17 and free micelles started to form. The authors verified that this visualization strategy is suitable for studying interactions between neutral polymers and surfactants.
Another work done by the same group is not in the category of self-assembly visualization by AIE, but the authors have exploited the high fluorescence quantum yield of TPE in micelles. They synthesized amphiphilic surfactants with TPE and a quaternary ammonium tail to build micelles as donor scaffolds for measuring the solubilization sites of solubilized substances. [52] When the substances as excitation energy acceptors were inserted into the micelles, TPE emission was weakened, accompanied by increased fluorescence signals of the solubilized substances due to FRET. By calculating the FRET distance between the anchored TPE and soluble acceptors, the location of solubilized substances in spherical micelles was accurately measured. In contrast to conventional methods, this measurement based on FRET does not rely on the micellar microenvironment. We present here this study for inspiring scientists to design simpler yet more powerful ways of "seeing" supramolecular assembly by rationally mixing different monomers in one micelle. This will lead to more abundant photophysical property changes during the self-assembly process for its real-time monitoring.
The Li group is interested in preparing bright AIE polymersomes due to their potential applications in cell imaging and drug delivery with high contrast. [91] In 2018, they synthesized an amphiphilic block copolymer (M19), where the hydrophobic block is a TPE-containing poly(trimethylene carbonate) ( Table 1). [92] M19 could form vesicles (SA19) in tetrahydrofuran (THF)/water mixtures. The corresponding fluorescence intensity evolution of M19 through the self-assembly process was studied. There are two main stages accompanied by abrupt fluorescence intensity changes: the first one was caused by the formation of polysomes (f w > 20%), which restricted the intramolecular motion of TPE. At this stage, THF still existed in the membrane of the vesicles. However, with the water content increasing (f w > 60%), osmotic pressure forced the organic solvent to be released, resulting in more effective aggregation of the TPE groups. This second stage could not be observed by regular microscopy analysis since the sizes and numbers of the formed polymersomes had already reached a plateau, thereby demonstrating the superiority of the AIE technique. It is believed that such AIE polymersomes with remarkable fluorescence changes by disturbing self-assembly processes may be used to monitor drug distribution and delivery in situ.
Later on, the same group reported nanoporous vesicles (E/Z-SA20) prepared from (Z)-and (E)-stereoisomers of an amphiphilic polymer containing hydrophobic TPE, cholesterol groups, and hydrophilic PEG chains (E-M20 and Z-M20, Table 1). [93] In acetone, both Eand Z-M20 were not emissive. However, their assemblies constructed in water showed bright cyan fluorescence, in which vesicles (E-SA20), cylindrical micelles (Z-SA20), and the nanoporous vesicles (E/Z-SA20) were formed from E-M20, Z-M20 and the mixtures of Eand Z-M20, respectively. Upon UV irradiation, the simultaneous (Z)-to (E)-and (E)-to (Z)photoisomerization caused continuous interchange of the hole parts and strand parts of E/Z-SA20, resulting in the vesicle membrane of E/Z-SA20 fluctuating. This process was also "seen" by epifluorescence optical microscopy because of the excellent photostability of the AIE polymersomes under high-power UV light (15 mW/cm 2 ).
To explore the hierarchical self-assembly process and uncover its underlying mechanism, the Tang group designed Au(I) building blocks S-M21 and R-M21 with AIE features (Table 1). [94] They are capable of self-assembly via inter-and intramolecular π-π stacking interactions, CH⋅⋅⋅F interactions, and weak Au-Au interactions to form vesicles (R/S-SA21). Time-dependent CD measurements suggested the dynamic morphological transitions of the self-assemblies, which were further verified by SEM, AFM, and TEM images. Unfortunately, no obvious circularly polarized luminescence (CPL) signals were observed during the self-assembly processes, presumably resulting from the low fluorescence quantum yields of the enantiomers. The authors finally realized CPL emitting by co-assembling typical luminogens with the above-mentioned chiral templates. This enables effective tuning of the luminescence properties of the supramolecular assemblies. In another work, Tang, Meng, Dang, and co-workers successfully visualized the formation and growth of self-assembling helixes of AIEgens by stimulated emission depletion nanoscopy. [95] However, no chiral signal was detected in this study. Thus, in situ visualization of hierarchical self-assembly by aggregation-induced CPL remains pending and challenging.
Another work done by the Tang group revealed the fusion and fission of vesicles (SA22-1 and SA22-2) by AIE changes (Table 1). [96] M22 was a negatively charged molecule with eight carboxyl groups, which was able to self-assemble with cetyltrimethyl ammonium bromide (CTAB) and Fe 2+ to form vesicles (SA22-1) by coordination and electrostatic interactions. By means of redox treatments between Fe 2+ and Fe 3+ ions, the vesicles displayed reversible fusion and fission behaviors, accompanied by changes in size and Zeta potential. To be specific, when Fe 2+ was oxidized to Fe 3+ , most of the Fe 3+ ions formed hydrates that lowered the electrostatic repulsion and decreased the vesicle curvature, thereby generating large vesicles (SA22-2) via fusion of SA22-1, which was reversible by reducing Fe 3+ to Fe 2+ . To visually monitor the changed molecular packing during the vesicle fusion and fission, Co 2+ ions were employed to avoid the fluorescence quenching caused by Fe 2+ and Fe 3+ ions. The M22@8CTAB vesicles have bright fluorescence. When Co 2+ ions were added gradually, the fission process took place and induced a decreased fluorescence intensity of vesicles accompanied by a hyperchromatic shift from 486 to 455 nm. This was attributed to the loosely arranged AIEgens in the fission assemblies. On the contrary, as ethylenediaminetetraacetate (EDTA) was added to remove the charges in the membrane of Co 2+ @vesicles, the fusion of the vesicles recovered the fluorescence signals of M22@8CTAB with redshift from 455 to 488 nm due to the closer packing of the AIEgens.
It was found that the AIE-active amphiphilic copolymers M23 could form vesicular structures in water by using THF as the cosolvent (SA23-2). [97] However, M23 was self-assembled into micelles (SA23-1) in N,Ndimethylformamide (DMF)/water mixtures (Table 1). This was attributed to the different solubility of the anthracene core in different organic solvents. The lower solubility of anthracene in DMF facilitated its compact molecular packing, leading to the significantly higher fluorescence intensity of the micelles than vesicles formed in the THF/water mixtures. This work corroborated the feasibility of visualizing different supramolecular assembly morphologies of a monomer via changed AIE signals.

VISUALIZATION OF THE SUPRAMOLECULAR POLYMERS
Supramolecular polymers are typical self-assemblies with polymeric characteristics. [98][99][100][101] On account of the dynamic nature of the supporting noncovalent interactions, supramolecular polymerization occurs with reversible properties and structures, which offered opportunities for investigating their dynamic processes by the variation of AIE signals. [102,103] This can be further applied to visualize the responses of supramolecular polymeric materials to some stimuli, resulting in the detection of isomer recognition, chiral transition, metal ion detection, target imaging, polymerization degree, and so on.
Efficient separation and photoisomerization of stereoisomers are of vital importance to life and material science. The Tang group distinguished (Z)-and (E)-isomers and monitored their photoisomerization by AIE techniques. In 2017, they obtained pure stereoisomers of ureidopyrimidinone (UPy)-functionalized tetraphenylethenes (TPE) (Z-M24 and E-M24), which were synthesized by McMurry coupling and isolated by simple column chromatography ( Figure 7A). [104] It was demonstrated that Zand E-M24 possessed AIE properties and supramolecular polymerizabilities. In the self-assembly state, their different molecular configurations resulted in distinguishable fluorescence colors. To be specific, the (Z)-assemblies were green-emitting, while the (E)-cousins showed blue emission. In 2019, the same group employed AIE characteristics and supramolecular polymerization of Zand E-M24 for control and visualization of the photoisomerization process ( Figure 7B). [105] In chloroform with a trace amount of trifluoroacetic acid (CF 3 COOH), an SA24-1, SA24-2, and SA24-3. Reproduced with permission. [104] Copyright 2017, The American Chemical Society. (B) Schematic representation of regulating the photoisomerization yield of Z-M24. Reproduced with permission. [105] Copyright 2019, The American Chemical Society. (C) Chemical structures of M25 of different chirality and geometry, as well as schematic representation of the corresponding supramolecular polymers. (D) Fluorescence spectra changes of M25 upon excitation at 312 nm (up) and 365 nm (bottom). Reproduced with permission. [54] Copyright 2022, The American Chemical Society. (E) Chemical structures of M26-1 and M26-2, as well as schematic illustration showing the detection and removal of Hg 2+ by the supramolecular polymers (SA26-1). Insets: photos of detection and removal of Hg 2+ by SA26, as well as plot of fluorescence intensity of SA26-1 at 388 nm against the concentration of Hg 2+ . Reproduced with permission. [106] Copyright 2019, The American Chemical Society equivalent amount of (Z)-and (E)-isomers was formed after the photoisomerization of Z-M24 because their absorption spectra resemble each other, and intermolecular hydrogen bonds were destructed by the organic acid. However, Z-M24 in chloroform could be transformed to the (E)-isomer with a high reaction yield of 68.1%. This was attributed to the higher supramolecular polymerizability of E-M24, which hampered the backward reaction to a certain extent.

F I G U R E 7 (A) Chemical structures of Z-M24 and E-M24, as well as schematic representation of
In this way, the isomerization reaction yield of Z-M24 could reach 100% in THF, which served as a good solvent for the (Z)-isomer assemblies but could not effectively dissolve the (E)-counterpart. During the Z-M24 photoisomerization, the polymeric aggregates of (E)-isomers formed with bright AIE signals. Therefore, this process could be observed by the fluorescence color changes from weak green to bright blue. The reduced solution transparence in the photoisomerization process in THF provided opportunities to develop optical power limiting materials.
Chirality, as another form of stereoisomerism, a transformation from the molecular to macroscopic level is far from fully understood. The Feringa group fabricated supramolecular polymers from the molecular motor (P,P)-cis-M25, which could exhibit another three states ((M,M)-trans-M25, (P,P)-trans-M25, and (M,M)-cis-M25) with distinct geometries and chiralities during the unidirectional rotation ( Figure 7C). [54] On this basis, the helical fibers of (P,P)-cis-M25 supramolecular polymers were completely transformed to (P,P)-trans-M25 micelles by irradiating with light for 2 min and then staying in the dark for 10 min. The conversion of (P,P)-trans-M25 to metastable (M,M)-cis-M25 was performed by the subsequent irradiation at 312 nm for 10 min, leading to a photostationary state with a considerate amount of (P,P)-trans-M25 remained. Hence, the micelles could not be restored to helical fibers, but rather worm-like fibers, when the obtained (M,M)-cis-M25 was transformed to the original state of the molecular motor. Gratifyingly, during the changes of the chirality and geometries of M25, the corresponding supramolecular assemblies also displayed variable AIE properties ( Figure 7D). Upon the excitation at 312 nm for 2 min, the AIE intensity of the helical supramolecular polymers decreased and redshifted from 440 to 480 nm owing to the transition of (P,P)-cis to (M,M)-trans-M25.
Keeping the water solution in the dark for 10 min, the generation of (P,P)-trans-M25 resulted in the fluorescence increase at 440 nm. When the (P,P)-trans-M25 micelles were irradiated to the photostationary state, the emission decreased and redshifted again due to the production of (M,M)-cis-M25. These AIE signal changes should be attributed to the different molecular configurations and packing of M25 in supramolecular assemblies. The remarkable AIE differences were also observed upon excitation of the sample solution at 365 nm ( Figure 7D). This work presents a facile way to reach a deeper understanding of the multistate stimuli-responsive materials.
Hg 2+ is regarded as one of the most detrimental environmental pollutants and can seriously damage the health of human beings. The development of a comprehensive strategy for the real-time detecting and rapidly removing Hg 2+ from water is quite important. In 2019, the Yang group constructed an AIE-based liner supramolecular polymer (SA26-1) based on host-guest interactions ( Figure 7E). [106] The [2]biphenyl-extended pillar [6]arene with two thymine sites (M26-2) served as the macrocyclic host and the TPE-bridged bis(quaternary ammonium) with AIE properties (M26-1) was the guest molecule. Because Hg 2+ ions can interact with thymine sites to form strong covalent bonds, the SA26-1 thus further self-assembled into spherical supramolecular nanoparticles (SA26-2) upon the addition of Hg 2+ . Accordingly, a remarkable fluorescence enhancement was observed due to the RIR of TPE. The "turn-on" AIE had a linear relationship with Hg 2+ in the concentration range of 0-15 μM, determining the detection limit to be 3 × 10 −7 M . In particular, Hg 2+ ions in the supramolecular polymers could be removed by Na 2 S and realized regeneration of the AIE probe.
Utilizing the AIE features, emission signals of supramolecular polymers could be tuned by controlling the reversible noncovalent interactions. On this basis, some related dynamic processes were visually tracked under time-dependent confocal microscopies. In 2021, Hyeon and co-workers introduced Zn 2+ ions to a nonemissive gold cluster (M27) by the coordination of Zn 2+ with carboxylate groups, resulting in a highly fluorescent gold cluster assembly ( Figure 8A). [107] Compared with M27, the gold supramolecular polymer (SA27) showed highly bright greenish-blue fluorescence with a fluorescence quantum yield reaching 90% under UV light excitation. This was attributed to a new radiative channel caused by special gold-gold interaction among gold clusters and the rigidified chemical environment induced by the coordination interaction. Such a mechanism for fluorescence originating from the metal-ligand supramolecular assembly was typical in elucidating AIE behaviors. Furthermore, upon the addition of EDTA, SA27 disassembled with gradual fluorescence disappearance, which could be recovered by adding more Zn 2+ ions. Benefiting from the visible fluorescence changes by controlling the formation and breakage of the coordination, the SA27 was successfully used as a trackable drug delivery system with biocompatibility and biodegradability. Under the time-dependent confocal microscopy, the release of doxorubicin was verified by observing the gradual dimming fluorescence of the supramolecular assembly.
Organelles are important components of cells. Its visualization is conducive to the understanding of various cellular processes. In 2018, the Liu group reported a two-stage supramolecular polymer (SA28-2) for lysosome-targeted cell imaging ( Figure 8B,C). [108] In the first stage, the binary supramolecular polymer (SA28-1) was fabricated by using an anthracyl pyridinium derivative (M28) and cucurbit [8]uril (CB [8]). The next stage is the interaction between SA28-1 and the amphiphilic sulfonatocalix- [4]arene (SC4AD) for forming nanoparticles. By introducing CB [8] and SC4AD, respectively, in the two stages, the supramolecular polymeric material with enhanced near-infrared (NIR) fluorescence was obtained. It was inferred that J-aggregation caused by CB [8] complexation and AIE activation by calixarene is responsible for the redshift from 625 to 655 nm and enhancement of fluorescence. The supramolecular polymeric nanoparticles successfully realized the lysosome-targeted imaging in cells.
Determining the degree of polymerization (DP) is of great significance to the structural characterization of supramolecular polymers. Recently, the Ji group reported an attractive strategy that molecular weight can be visually differentiated by fluorescence color variation of UPy-based supramolecular polymers ( Figure 8D). [109] The monomer (M29) was synthesized by decorating two UPy groups to a pyrene benzohydrazonate-based AIEgen. [110] It formed supramolecular polymers (SA29) through quadruple hydrogen bonding forces between two UPy groups. As the concentration of M29 increased from 0.1 to 100 mM, the average DP for SA29 raised from 1.00 to 293. Interestingly, the corresponding fluorescence colors varied from dark blue to yellow-green in the same concentration range due to the aggregation state changes of M29 at different concentrations ( Figure 8E). The relationship between DP and fluorescence color was thus established, and the visual distinction of molecular weight was realized ( Figure 8F). The work paves the way for the molecular weight visualization of various polymers with AIE characteristics.

VISUALIZATION OF THE SUPRAMOLECULAR GELS
Supramolecular gels exhibit a network structure that is composed of a dispersed phase and a dispersing medium based on noncovalent interactions. [59,[111][112][113][114] The supramolecular gels with AIE features are usually constructed by the selfassembly of AIEgens, which occurs aggregation in the gel matrix. Thus, the gelation process can be monitored by the changes in AIE signals. In addition, the sol-gel transition can be realized through external stimuli, which will be accompanied by changes in AIE properties, thereby visualizing the stimuli responsiveness.
In situ real-time monitoring of the entire gelation process contributes to a deeper understanding and better utilization of gels. In 2016, Wang, Nie, and colleagues first reported the visualization of gelation processes by using TPE-labelled chitosan (M30, Figure 9A). [115] For the M30 aqueous solution, no bright fluorescence was observed under the confocal laser scanning fluorescence microscope ( Figure 9B). However, during the thermal gelation process, the bright fluorescence appeared gradually and eventually reached a plateau. With the removal of LiOH and urea, the bright fluorescence areas fur-ther subdivided and contracted to form a reticular structure, which was the key step for forming the chitosan hydrogel. The changes in the fluorescence areas were consistent with the evolution of macroscopic characteristics of the gels during the gelation process. Combined with other pseudo in situ investigations, they clarified that the formation of junction points, including hydrogen bonds and crystallin, was the important driving force for the occurrence of two different gelation processes.
Stang et al. prepared a multifunctional metallacage-based supramolecular gel by the combination of orthogonal metal coordination and host-guest interactions for achieving the visualization of gel-sol transitions. [18] First, they fabricated a tetragonal prismatic metallacage (SA13-1) with four 21crown-7 units from cis-Pt(PEt 3 ) 2 (OTf) 2 (M7-4), TPE-based sodium benzoate ligands (M12-1), and linear dipyridyl ligands (M31-1) via the metal-coordination ( Figure 9C). SA13-1 was further assembled by a bisammonium linker (M31-2) to form the supramolecular gel (SA31-2). SA31-2 exhibited AIE properties due to the supramolecular gelation. On the other hand, SA31-2 could be transformed to the solution by heating or adding the potassium ions since the host-guest interactions of crown ethers and ammonium Reproduced with permission. [117] Copyright 2020, Wiley-VCH GmbH salts were weakened ( Figure 9D). This gel-sol process could be monitored by the fluorescence intensity changes, which decreased gradually and would return to the original state by cooling or adding excess 18-crown-6 (18C6).
In 2018, Ramström and co-workers observed that the enaminitrile-based derivatives could exhibit acid/baseresponded configuration transition with the AIE effect. [116] In this regard, they designed an enaminitrile-based switch (M32) by modifying a glutamic acid with long aliphatic chains as rotors ( Figure 9E). The sol-gel transition process could be visually monitored by the corresponding AIE fluorescence off-on effect. The E-isomer of M32 (E-M32) only formed a suspension in nonpolar solvents. Surprisingly, when methylsulfonic acid (MSA) was added, followed by heatingcooling, the suspension converted to the gel of the protonated Z-isomer (Z-M32-H + ) with a fluorescence change from weak green to bright blue ( Figure 9F). Besides, the formed gel rapidly disassembled when triethylamine (Et 3 N) was added to the system, and the entire sol-gel process could be cycled at least five times. This research also provided possibili-ties for the visual monitoring of pH changes in bioimaging, environmental analysis, etc.
Later, Xin et al. constructed metal-organic gels based on an Ag nanocluster (M33). They showed AIE characteristics and fluorescence to phosphorescence conversion behaviors by gelation ( Figure 9G,H). [117] M33 was prepared by adding silver nitrate and thiosalicylic acid in the alkaline aqueous solution and can display extremely weak photoluminescence at room temperature. Upon adding ethanol with the volume percentage of 70%, the aqueous solution of M33 (c M33 = 5.0 mmol/L) converted to gels with aggregation-induced bright yellow emission at 293 K. According to the SEM and TEM images, M33 self-assembled into highly ordered entangled fibers with tight cluster stacking to suppress the motions of each M33 in the gelation process ( Figure 9I). The authors inferred that the multiple intra-and intercluster interactions, especially the hydrogen bonds, favor the nanocluster-based AIE system. Interestingly, this system occurred fluorescence to phosphorescence transition in the gelation process, which was attributed to the structural TA B L E 2 Summary of the partial AIE-based supramolecular gels for visualization

No.
Name Chemical structure of monomer Visualization Ref.

M35
Detection of total proteins in gels [121] 3 P A S Shape deformation and pH stimulation [122] M36 4
The emission signal amplification and switching visualized the gelation process. Moreover, the morphologies and photoluminescence of the metal-organic gels could be regulated by changing the temperature from 293 K to 363 K, showing good reversibility and excellent linear relationship between them.
Fluorescence changes can also be achieved by doping chemical or biological species directly into the gel, which self-assembly with the gel, thus providing possibilities for monitoring. [118,119] Based on this, visual monitoring of specific species can be achieved.
Visual monitoring of the reaction rates favors understanding the photoreaction mechanism. The Tang group reported a visible and rate-controlled photodimerization system based on the AIE technique and host-guest chemistry. [120] They designed two porous cyclodextrin-based hydrogels (β-SA34 and γ-SA34) by the nucleophilic aromatic substitution of epichlorohydrin to the hydroxyl groups of cyclodextrin ( Table 2). The two hydrogels were immersed into the aqueous solution of a cyanostilbene derivative (M34) to construct different complexes (β-SA34⊃M34 and γ-SA34⊃M34) based on the host-guest interactions between cyclodextrin and M34. The γ-SA34⊃M34 displayed yellow emission at 554 nm, but β-SA34⊃M34 showed fluorescence at 515 nm. This difference is attributed to the formation of different host-guest complexes with molar ratios of 2:2 for γ-SA34⊃M34 and 1:1 for β-SA34⊃M34. When γ-SA34⊃M34 was irradiated by UV light at 365 nm for an hour, M34 occurred 100% photodimerization, yielding photodimers with a fluorescence color change from yellow to green. It was demonstrated that the formation of 2:2 host-guest complexes improved the ability of intermolecular photodimerization of M34 due to the closer distance of two double bonds in separate molecules. In contrast, the fluorescence of β-SA34⊃M34 has no change after photoirradiation for 10 h because of the 1:1 host-guest assembling ratio.
Due to the special biological affinity and chromogenic reduction of silver ions, the chromogenic visualization of classical silver stains plays an important role in in-gel protein detection. However, there are many limitations, such as run-to-run variability, background staining, and quantification difficulty. On this basis, Tang, Chen, and colleagues suggested a novel fluorogenic visualization of silver ions based on the AIE behavior triggered by tetrazolate-Ag + interactions (Table 2). [121] They synthesized the AIEgen M35 as a water-soluble fluorogenic Ag + probe with invisible fluorescence in aqueous solution. By binding with Ag + , M35 can form aggregates and display bright green fluorescence. Combining the silver staining with the AIE Ag + probe, the authors achieved highly sensitive chromogenic visualization of in-gel protein with excellent linearity for protein quantification.
Later on, the Tang group reported a bioinspired hydrogel that can realize simultaneous changes in the fluorescence color, brightness, and shape deformation under pH stimulation. [122] This hydrogel was produced by encapsulating the aggregates of tetra-(4-pyridylphenyl)ethylene (M36) into the bilayer hydrogel actuators (poly(acrylamider-sodium 4-styrenesulfonate), PAS) via an asymmetrical distribution (Table 2). In water, the hydrophobic M36 formed aggregates with bright sapphire fluorescence. As the pH decreased, M36 was transformed to a hydrophilic tetraprotonated derivative, causing the fluorescence redshift and attenuation. However, when M36 was packed into PAS, the redshifted fluorescence of the hydrophilic tetra-protonated derivative was improved since the electrostatic interactions between the tetra-protonated derivative and the benzenesulfonate groups of PAS restricted the intramolecular rotations. Meanwhile, by crosslinking the tetra-protonated derivative, the active layer of the hydrogel started to shrink, and the shape of the actuator gradually transformed with different bending angles. Immersing the hydrogel in different pH solutions for 17 h, its fluorescence gradually redshifts with a shape transformation. Additionally, the same transformation of fluorescence and shape occurred when the hydrogel was immersed in pH 3.12 aqueous solution with different times. Thus, the shape deformation of the system could be visualized in situ by the variation of AIE characteristics.
In 2021, the same group suggested a strategy to implement remote spatio-temporal control of luminescence in a non-contact way. [123] A fluorescent gel with UV-mediated fluorescence changes was fabricated based on a TPEcored salicylaldehyde compound (M37-1) and a linear acylhydrazine-terminated PEG chain (M37-2), which can react to form salicylaldehyde benzoylhydrazone units as coordination sites for metal ions ( Table 2). The ligand units effectively responded to Al 3+ , Zn 2+ , and Cd 2+ ions to switch the fluorescence of the gel. When the aqueous of Al 3+ , Zn 2+ , and Cd 2+ was coated to the surface of the fluorescent gel, respectively, its fluorescence intensity was improved and the fluorescence color varied from blue, green to yellow. The mechanism behind the fluorescence change was explored by studying a modal compound, TPE-based salicylaldehyde benzoylhydrazone multi-armed AIEgen (TPE-4SAH). There is only a negligible fluorescence change upon adding the metal ions (Al 3+ , Zn 2+ , and Cd 2+ ) into the solution. However, obvious fluorescence changes were observed when the solution of TPE-4SAH and metal ions was irradiated by UV light. Under the excitation of UV, the deprotonation of the phenolic proton occurred more easily in the excited state, which caused charge separation of ligands and endowed them with superior ability to coordinate metal ions. Thus, the phototriggered AIE phenomenon is presumably attributed to the transformation from three-coordinated complex to six-coordinated complex, which resulted in the formation of larger and tighter aggregates to suppress the non-radiative decay.
Tracking the fluorescence changes is very appealing for chiral recognition and separation due to its simplicity and high efficiency. Recently, Tang et al. designed two TPE-based chiral AIEgens with two optically pure 1-cyclohexylethylamine units (R-M38-1 and S-M38-1) ( Table 2). [53] R-M38-1 showed high enantioselectivity for D/L-Boc-glutamic acid (D/L-M38-2). In 1,2-dichloroethane, R-M38-1 is able to enantioselectively recognize D-M38-2 to generate a suspension and eventually convert to a transparent gel after standing for several hours. Both the suspension and gel can generate bright green fluorescence. The fluorescence intensity of the formed AIEgen/acid complexes reached 103 times higher than R-M38-1. However, the mixture of R-M38-1 and L-M38-2 only resulted in clear solution with inapparent emission. The authors speculated that the chargeaided hydrogen bonding interactions between the amino groups of R-M38-1 and the negative D-M38-2 effectively restricted the free phenyl ring rotations of TPE, thus leading to strong fluorescence.

SUMMARY AND OUTLOOK
In this review, we have summarized the recent advances of monitoring the formation of supramolecular assemblies and their further dynamic behaviors by AIE techniques. The organization is based on the types of supramolecular assemblies, including metallacycles/cages, micelles/vesicles, supramolecular polymers, and supramolecular gels. In essence, self-assembly brings the building blocks together, which establishes the connection between AIE and supramolecular chemistry. Changes in molecular arrangements and morphologies of the supramolecular assemblies result in different AIE signals with the advantages of signal amplification and low background noises. For these reasons, the marriage of AIE and supramolecular assembly has led to an easy, simple, and in situ approach to the supramolecular assembly visualization. For example, the assembly process, morphological transformation, drug delivery, and stimuli responsiveness have been observed by the changes of AIE characteristics. It is clear that the visualization of dynamic processes associated with supramolecular assembly has made attractive progress based on AIE properties. However, compared with other characterization techniques, the development of this area is still in its infancy, thus existing challenges and opportunities. First, TPE, as the typical AIEgen, plays a major role in the visualization of supramolecular assembly. In fact, many other AIEgens, including triphenylamine, heteroatom-bridged cyclopentadienes, Schiff bases, etc., have been developed, which exhibit great potential in biological imaging, chemical sensing, stimuli responsiveness, etc. We infer that associating supramolecular chemistry with more types of AIEgens for the visualization of supramolecular assembly-related behaviors deserves to be further explored. Second, the magnitude of the AIE intensity changes is a key parameter for studying the dynamic processes related to supramolecular assembly. Insignificant changes will limit the accuracy of the characterization. Thus, in addition to increasing the AIE intensity differences before and after supramolecular assembly, more parameters, that is, color, lifetime, phosphorescence, and others, can be involved in characterizing the supramolecular assembly-related process for more accurate and multi-dimensional visualization.
In addition, most recent developments in supramolecular assembly visualization by AIE only concern visible fluorescence changes by the naked eye. These provide clues for specific self-assembly stages, such as the critical micelle concentration. However, accurate quantification of the selfassembly degree by AIE is a lot more difficult, and different AIE probes may lead to different results. It is anticipated that a standard AIEgen of known quantity of fluorescence in different aggregation degrees can be employed for measuring different supramolecular assemblies. Fortunately, there are clear signs that recent activities have been geared toward bridging the gaps between visualization and direct quantification by AIE signals. For example, the Tang group has recently achieved the quantitative detection of sodium dodecylbenzenesulfonate through the co-assembled micelles, [88] the Yang group quantitatively detected Hg 2+ via supramolecular polymerization, [106] and the Ji group bridged the degree of polymerization and fluorescence colors by AIEgen-based supramolecular polymers. [109] These advances pave the way for quantitative visualization in terms of higher sensitivity, selectivity, and real-time detection.
Besides the broad interest in visualizing the in vitro biological processes by AIEgen-based supramolecular assemblies, there are few researches that focus on the in vivo monitoring of supramolecular assemblies, which may shed some light on the disease and biomimetic materials. The inherent responsiveness of self-assemblies, and their abilities to regulate materials features (i.e., toxicity and hardness) with AIE signal changes are appealing. We predict that the utilization of supramolecular assembly visualization will lead to advances in disease diagnosis and treatment.
In conclusion, the visualization of supramolecular assembly based on AIE characteristics is still a young field and there is a broad scope that can be further explored. With continuous efforts, we believe that the study of selfassembly by AIE will lead to a deeper understanding of its mechanisms and structure-function relationships, which will make supramolecular assemblies more appealing in various applications. At the same time, we expect that this review will bring benefits to the development of visualization of supramolecular assembly in the near future and thus make a contribution to this field.

A C K N O W L E D G M E N T S
Hui-Qing Peng thanks the National Natural Science Foundation of China (22105016 and 22005195) and the Ministry of Science and Technology of China (2022YFA1505900). Hui-Qing Peng is also grateful for support from the Open Fund of Guangdong Provincial Key Laboratory of Luminescence from Molecular Aggregates, South China University of Technology (2019B030301003). Bin Liu is grateful for financial supports from the National Natural Science Foundation of China (52002015 and 22275010) and the Fundamental Research Funds for the Central Universities (buctrc202006).

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.