Near-infrared II (NIR-II) microscopy, which enables in vivo deep-tissue visualization of vasculature and cell activities, has been a promising tool for understanding physiological mechanisms. However, the volumetric imaging speed of the NIR-II microscopy is hindered by scanning strategy, causing limitations for observing instantaneous biological dynamics in 3D space. Here, we developed a NIR-II light-field microscopy (LFM) based on selective-illumination and self-supervised implicit neural representation (INR)-reconstruction, which allows ultra-fast 3D in vivo imaging (100 volumes/s). Through integrating INR with view-wise aberration correction, we could conquer the artifacts induced by the angular subsampling and refractive index variation, achieving single-cell resolution at a volume of 550 & micro;m diameter and 200 & micro;m thickness. The volumetric selective-illumination overcomes the influence of out-of-focus background, together with the low scattering advantage of NIR-II wavelength, extending the imaging depth to 600 & micro;m. The developed aberration-corrected implicit neural representation reconstruction (AIR) NIR-II LFM showcases its capability by monitoring hemodynamics of mouse brain under norepinephrine and flow redistribution of ischemic stroke in 3D vascular network, as well as noninvasively tracking immune cell activities inside subcutaneous solid tumor through intact skin. This approach represents a significant advancement in 3D in vivo imaging, holding great potential in biomedical research and preclinical studies.

ABSTRACT The resulting product of biocoupling is a bioconjugate, typically formed by linking molecules to proteins, oligosaccharides, nucleic acids, or synthetic polymers. By conjugating fluorescent probes with bioactive molecules via click chemistry or bioorthogonal reaction, functional materials with high specificity, sensitivity, and accuracy can be developed. A particularly promising strategy involves the use of aggregation‐induced emission (AIE) fluorophores, which exhibit augmented luminescence intensity and excellent photostability when in the aggregated state, making them especially suitable for bioimaging and biosensing applications. The rapid expansion of AIE active bioconjugates now calls for a comprehensive review to summarize and systematize recent advances. In this review, we direct our focus toward the biosensing, bioimaging, and therapeutic applications of AIE active bioconjugates prepared via click chemistry or bioorthogonal reactions. We anticipate that this overview will promote the development of versatile AIE bioconjugates and inspire further innovations in bioorthogonal chemistry for biomedical applications.

ABSTRACT Multimodal phototheranostics, capitalizing on the synergistic interplay of distinct optical diagnostic and therapeutic modalities, stand at the forefront of modern theranostic development. Among the pursued approaches, the “one‐for‐all” paradigm, which utilizes a single organic component to achieve four classic phototheranostic functions (i.e., fluorescence imaging, photoacoustic imaging, photodynamic therapy, and photothermal therapy), has gained considerable traction. Benefitting from the simple building block, the “one‐for‐all” phototheranostic agents generally exhibit well‐defined molecular architectures, finely tunable excited‐state energy pathways, excellent batch‐to‐batch reproducibility, predictable pharmacokinetic profiles, and superior biocompatibility, collectively pointing toward strong clinical translation potential. In this context, aggregation‐induced emission luminogens (AIEgens) have arisen as an exceptionally versatile molecular template, because their unique structural attributes offer precise control over the aggregation behavior and photophysical dynamics required for integrated phototheranostic design. In view of this, this review aims to propose a consensus that AIEgens serve as a quintessential template for “one‐for‐all” multimodal phototheranostics, and articulate the underlying rationale from a photophysical perspective. It comprehensively examines molecular engineering principles and performance optimization tactics for AIEgen‐based “one‐for‐all” systems, framed within the fundamental photophysical concept of excited‐state energy regulation. Furthermore, with a translational outlook, this review critically assesses the current challenges and proposes forward‐looking research trajectories, seeking to identify promising, underexplored avenues that could accelerate practical clinical application.

The integration of aggregation-induced emission (AIE) luminogens (AIEgens) with hydrogel materials has significantly expanded their applications in precision medicine, fostering groundbreaking innovations in biomedicine. AIEgens exhibit exceptional photostability, a high signal-to-noise ratio, and stimuli-responsive properties making them uniquely suited for constructing theranostic platforms for image-guided diagnosis and therapy. Meanwhile, hydrogels provide an ideal matrix for precision diagnostics and therapeutics due to their outstanding biocompatibility, tunable physicochemical properties, and capacity to serve as carriers for drugs and molecular probes. Given the immense potential of AIEgen-hydrogel composite systems in precision medicine, this review systematically summarizes recent advances in their applications, including cancer-targeted therapy, biofluorescent probes, antibacterial treatment, biomimetic cell culture, biomarker detection, and drug delivery/release monitoring. Furthermore, we critically analyze the key challenges hindering their widespread adoption and discuss future research directions. Together, AIEgen-integrated hydrogel systems represent a transformative approach in precision medicine, driving significant progress through their versatile biomedical functionalities.

Reactive oxygen species (ROS)-assisted photodynamic antibiosis emerges as an effective non-antibiotic way to eradicate bacteria in infected wounds. To minimize the indiscriminate damage of excessive ROS to normal tissues, on-demand release of photosensitizers (PSs) is highly desired. Herein, we report a novel aggregation-induced emission (AIE) PSs-loaded hydrogel wound dressing by incorporating AIE PSs into hydrogels prepared from sodium alginate and hyaluronic acid (HA) upon crosslinking in Ca2+ solution. By virtue of the hyaluronidase (HAase) that is secreted from bacteria, AIE PSs can be released from hydrogels due to the HA degradation. Particularly, the HAase activity can be elevated in the presence of low concentrations of Ca2+, where HAase tends to adopt a more stable conformation, whereas high concentrations of Ca2+ triggers the salt precipitation of HAase, thereby inhibiting its activity. Benefitting from this advantageous Ca2+-dependent HAase responsiveness, on-demand release of AIE PSs regulated by the Ca2+ that freely diffused from hydrogels is actualized, as evidenced by the accelerated healing of in vivo bacteria-infected wounds. Overall, this work not only reports the bidirectional regulation mode of Ca2+ on HAase activity and elucidates the mechanism for the first time, but also provides a new strategy with on-demand release of antibacterial PSs for accelerated wound healing.

Fungal biofilm infections pose a serious and growing threat to human health. To address this challenge, we developed a multifunctional nanoplatform (ASE@B NPs) that integrates a second near-infrared (NIR-II) photothermal agent, biofilm-targeting anti-microbial function, and photothermally triggered nitric oxide (NO) release. This system enables precise targeting of the infected microenvironment through a carrier containing acid-responsive segments, thereby achieving combined anti-fungal and anti-inflammatory effects against fungal biofilms. A key to this advance was the enhancement of photothermal conversion efficiency (PCE) via molecular engineering of aggregation-induced emission (AIE)-active NIR-II luminogens. By shifting the donor-acceptor-donor (D-A-D) configuration from symmetric to asymmetric, we successfully converted their emission behavior from aggregation-caused quenching (ACQ) to AIE. Furthermore, by leveraging the acidic biofilm microenvironment and negatively charged surfaces of fungal cells, we employed an amphiphilic polymer carrier incorporating a weak acid-responsive charge-reversal moiety. This carrier was coloaded with a photothermal-responsive NO prodrug (BNN6) and the AIE luminogen. The resulting nanoplatform exhibits a negative surface charge under physiological conditions, ensuring prolonged blood circulation and biosafety, while switching to a positive charge in acidic biofilms to promote penetration and fungal binding. Collectively, this multifunctional nanoplatform enables efficient biofilm disruption and microenvironment reprogramming, offering a promising theranostic strategy for combating drug-resistant fungal infections.

Three-photon microscopy (3PM) utilizing the near-infrared-III (NIR-III) window (1600–1840 nm) provides a pivotal tool for high-resolution visualization of cerebral vasculature in vivo, owing to its superior deep-tissue penetration capability. However, imaging quality is critically dependent on the performance of fluorescent probes. This study proposes a probe optimization design strategy evolving from mirror symmetry to centrosymmetry, leading to the synthesis of a highly symmetrical aggregation-induced emission (AIE) molecule, T4PQ. The centrosymmetric structure enables precise matching of donor (D)-acceptor (A) units, facilitating uniform electron cloud delocalization and directional charge transfer. This enhances exciton formation efficiency and suppresses non-radiative decay, thereby improving the fluorescence quantum yield. Furthermore, the highly symmetrical configuration significantly boosts the three-photon absorption cross-section by promoting electron delocalization and increasing the transition dipole moment, enabling stronger nonlinear optical responses under long-wavelength excitation. As a result, the designed T4PQ nanoparticles achieve optimized probe performance, combining an enhanced three-photon absorption cross-section, high fluorescence quantum yield, and excellent photostability. The corresponding nanoparticles (T4PQ NPs) exhibited a significantly enhanced three-photon absorption cross section, high fluorescence quantum yield, and excellent photostability. In murine in vivo experiments, T4PQ NPs achieved three-dimensional cerebrovascular imaging at a depth of 1785 µm and enabled real-time dynamic observation of hemodynamics in microvessels at a depth of 1006 µm, while demonstrating good biocompatibility. This research validates the advantage of centrosymmetric molecular structures in the design of deep-brain imaging probes, providing a high-performance tool for neurovascular research.

Chemiluminescence (CL), an excitation-free optical phenomenon arising from chemiexcitation during chemical reactions, has garnered considerable attention in biological sensing and imaging field. Occurring spontaneously, CL eliminates the need for real-time excitation, thereby mitigating issues like tissue autofluorescence, light scattering, and attenuation. Consequently, CL offers exceptional sensitivity, high signal-to-background ratio (SBR), and deep tissue penetration. However, conventional CL agents typically operate in the UV–visible region, limiting their bioimaging effectiveness due to undesired tissue contrast and insufficient penetration depth. Shifting CL spectrum to the near-infrared region (NIR, 650–1700 nm) would significantly enhance in vivo lesion visualization by providing improved sensitivity, SBR, and tissue penetration, facilitating more accurate disease diagnosis. Integrating NIR aggregation-induced emission luminogens (AIEgens) with CL represents a promising strategy, taking full advantage of the exceptional photophysical properties of AIEgens (including high brightness, large Stokes shifts, and broad spectral tunability) and the inherent merits of CL. This review comprehensively examines state-of-the-art AIE-enhanced NIR CL systems based on diverse substrates such as dioxetanes, peroxyoxalates, and luminol derivatives. We elucidate fundamental design principles, underlying luminescence mechanisms, and cutting-edge applications in bioimaging and theranostics. Finally, we critically analyze current challenges and future opportunities for advancing this technology toward clinical translation and precision medicine applications.

ABSTRACT “One‑for‑all” phototheranostics has emerged as a promising alternative to conventional cancer therapies. Aggregation‑induced emission luminogens (AIEgens) are exemplary templates for constructing such versatile systems. However, developing robust NIR‑II (1000–1700 nm)‐emitting “one‑for‑all” phototheranostic AIEgens remains challenging, due to the drastically lowered reactive oxygen species (ROS) generation capability and strong intermolecular π‐π stacking in the NIR‑II range. To address this, an ingenious dual‑acceptor strategy was proposed. It leverages the steric hindrance between two moderate‑strength acceptors to create a twisted dual‑acceptor configuration, which not only circumvents the detrimental effects caused by large planar strong acceptors, such as π–π stacking‐induced emission quenching and excited‐state energy loss, but also favors AIE nature. Moreover, moderate‑strength acceptors are readily functionalized, facilitating the introduction of substituents that enhance ROS performance. Through combining a strong donor, an optimized AIE small molecule, TTBI, was identified, which exhibits desirable NIR‑II emission, excellent type I ROS production, and a high photothermal conversion after formulation into nanoparticles (NPs). Coupled with outstanding stability and biocompatibility, TTBI NPs demonstrated exceptional 808 nm‑excitable NIR‐II fluorescence–photoacoustic–photothermal trimodal imaging and synergistic photodynamic–photothermal therapy in mouse orthotopic breast cancer models. This work provides new insights for creating superior NIR‑II multimodal phototheranostic agents.

Effective management of diabetic wound healing remains challenging due to the complex wound microenvironment, characterized by persistent infections and inflammation, as well as obstacles in tissue repair, including impaired extracellular matrix (ECM) remodeling and re-epithelialization. Herein, we propose a sequential integrating microenvironment regulation and re-epithelialization promotion (SIMREP) strategy based on zinc-doped glycyrrhizinate carbon dots loaded with aggregation-induced emission luminogens (AIEgens), namely TTPy-Zn@GCDs for accelerated diabetic wound healing. TTPy-Zn@GCDs effectively inhibit bacterial infection through AIEgen-based photodynamic therapy and promote the M2-phenotype polarization of macrophages by eliminating reactive oxygen species. Remarkably, TTPy-Zn@GCDs not only present a superior ECM remodeling ability by suppressing the overexpression of matrix metalloproteinase (MMP)-9 in diabetic wounds, but also promote keratinocyte migration and accelerate the re-epithelialization process by activating the hypoxia-inducible factor-1α (HIF-1α) signaling pathway. Together, the SIMREP strategy offers a promising approach for accelerating the healing process of infected diabetic wounds.

Photoimmunotherapy offers precise tumor ablation and systemic immunity through immunogenic cell death and cytotoxic T-cell activation. Among immunotherapies, antigen-specific vaccines targeting well-defined tumor antigens are particularly promising for personalized cancer treatment due to their durable immune surveillance and precise activation with minimal off-target effects. However, their integration with phototherapy remains almost unexplored. Herein, we report a synergistic strategy combining an antigen-specific vaccine with phototherapy. Specifically, motivated by the advantageous merits of multimodal phototheranostics, a multimodal phototheranostic agent (TBBSD) was rationally designed as the phototherapy module, while the hepatocellular carcinoma (HCC)-specific antigen glypican-3 (GPC3) protein and adjuvant cyclic diadenylate (c-di-AMP) were employed as the antigen-specific vaccine. By means of the complementary advantages, this photoimmunotherapeutic strategy achieves efficient ablation of primary tumors, simultaneously induces a robust systemic immune response, and fosters long-term immunological memory, which synergistically prevents tumor recurrence and suppresses the growth of both distant and pulmonary metastatic lesions. Collectively, these results underscore the therapeutic reliability and translational potential of this efficacious photoimmunotherapy strategy for precision cancer treatment.

The robust outer membrane (OM) barrier is a major contributor to antibiotic resistance in multidrug-resistant Gram-negative bacteria (MDR GNB). Disrupting this barrier presents a promising strategy to overcome this challenge. Herein, we propose a carbon monoxide (CO)-driven cascade inhibition strategy to disrupt the OM barrier, aiming to significantly boost the antimicrobial efficacy of existing treatments. As a proof of concept, we developed AIE&CO@G3, a nanogel that combines CO-releasing molecules (CORM-401) and aggregation-induced emission (AIE) photosensitizer (PSs). CO significantly potentiated the antimicrobial activity of AIE PSs-based antimicrobial photodynamic therapy (AIE-aPDT), with similar synergistic effects observed when combined with multiple first-line antibiotics. Mechanistically, CO-induced OM disruption facilitated the penetration of AIE PSs and antibiotics, thereby substantially boosting their efficacy both in vitro (against multiple MDR GNB) and in vivo (in models of MDR P. aeruginosa-infected bacterial keratitis and pneumonia). This was achieved by inhibiting adenosine triphosphate (ATP) synthesis and disrupting the biosynthesis and transport of glycerophospholipids (GPL) and lipopolysaccharides (LPS). This pioneering study highlights CO’s potential in OM disruption and provides a novel strategy for combating MDR GNB infections.

Sonodynamic therapy (SDT), as a promising noninvasive therapeutic modality with superior penetration depth, receives tremendous attention. To date, the widely accepted mechanism for reactive oxygen species (ROS) generation in SDT involves acoustic cavitation-triggered sonoluminescence (SL), followed by the SL-activation of sonosensitizers. However, current research on sonosensitizer development primarily focuses on promoting SL-to-ROS conversion, overlooking the essential role of the cavitation process. To fully unleash the potential of SDT, herein, a dual-enhanced strategy that harnesses the enhanced cavitation for SL generation and efficient SL-to-ROS conversion is developed for the first time to realize an all-around enhancement of SDT. Specifically, the proposed nano-sonosensitizer, namely MeTTh-PAE NPs, is released as hydrophobic aggregates with a rough surface in response to an acidic environment, allowing for highly enhanced cavitation-triggered SL under ultrasound. Meanwhile, as a typical aggregation-induced emission molecule, MeTTh demonstrates a highly promoted intersystem crossing process at its aggregated state, facilitating efficient SL-to-ROS conversion. Notably, combining these two fascinating attributes in MeTTh-PAE NPs results in an excellent sonodynamic antitumor effect in both in vitro and in vivo. This work proposes a novel strategy to fully exploit the potential of SDT and provides valuable insights for advancing the design of nano-sonosensitizers.

Angiogenesis is crucial for diabetic wound healing by facilitating the delivery of oxygen and nutrients. However, some challenges including bacterial susceptibility, inflammatory storm, and vascular regulatory factors deficiency persist. Herein, we develop an engineered aggregation-induced emission luminogens (AIEgens)-based framework with carbon monoxide (CO)-releasing property (named as T/MnCO@AMOF) for accelerating diabetic wound healing via simultaneously regulating the microenvironment and angiogenesis. T/MnCO@AMOF is constructed by co-loading AIE photosensitizer (TTI-COOH) and a CO generator manganese carbonyl (MnCO) within 1,1,2,2-tetra(4-carboxylbiphenyl)ethylene (TCBE)-based frameworks. On the one hand, T/MnCO@AMOF eliminates bacteria via zinc and AIE photosensitizer-based photodynamic therapy (PDT). On the other hand, the released MnCO from T/MnCO@AMOF scavenges excess endogenous hydrogen peroxide (H2O2) through Fentonlike reaction, and the generated CO polarizes macrophages from the pro-inflammatory M1 to the antiinflammatory M2 phenotype. Subsequently, the macrophage-derived vascular endothelial growth factor (VEGF) and the CO-activated hypoxia-inducible factor-1 alpha (HIF-1 alpha) pathway further stimulate angiogenesis. In order to enhance tissue penetration, a hyaluronic acid (HA)-based degradable microneedle (MN) patch is developed for targeted wound delivery. Impressively, the in vivo experiments confirm that T/MnCO@AMOFloaded MN exhibits potent performance of accelerating diabetic wound healing, following significant blood vessel regeneration. Taken together, the proposed multifunctional AIE MOF-based strategy provides a promising reference for high-quality restoration of diabetic wounds.

ABSTRACT The vasculature, as the essential biological network for oxygen and nutrients delivery and the dynamic regulatory center for physiological processes, is fundamentally important for maintaining human health and life quality. Accurate visualization of vascular structures, as well as real‐time monitoring of hemodynamic parameters and molecular profiles associated with vascular function, are therefore crucial for early diagnosis and preventive interventions of vascular diseases. Fluorescence imaging technology, particularly in the second near‐infrared window (NIR‐II; 1000–1700 nm), offers distinct advantages for these demanding imaging requirements not only due to its high sensitivity, excellent spatial resolution, and real‐time monitoring capability but also thanks to the superior signal‐to‐background ratio and large tissue penetration depth of NIR‐II fluorescence. Among diverse NIR‐II fluorescent probes, aggregation‐induced emission luminogens (AIEgens) stand out for their intrinsic organic nature and, more importantly, for their unique aggregation‐enhanced emission properties, which clearly differentiates them from traditional fluorophores and enable high‐resolution imaging. Currently, a series of high‐performance NIR‐II AIEgens featuring relatively high fluorescence brightness and long emission wavelengths with emission tails even extending into the NIR‐IIa (1300–1400 nm) and NIR‐IIb (1500–1700 nm) subwindows have been reported and demonstrated encouraging results in intravital fluorescence angiography. This minireview summarizes recent advances in NIR‐II AIEgens for various vascular imaging applications, categorized by anatomical locations, including cerebral, abdominal, hindlimb, ear, axillary, renal, and tumor angiography. The molecular design strategies and nanoengineering approaches to achieve longer emission wavelengths, higher fluorescence brightness, and improved bioavailability are highlighted. Finally, the remaining challenges and future directions are discussed from the aspects of materials engineering, application scenarios expansion, and clinical translation.

Fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) has emerged as a powerful tool for in vivo bioimaging, offering deep-tissue penetration alongside high spatiotemporal resolution and contrast. Owing to structural advantages, aggregation-induced emission (AIE) luminogens are recognized as an ideal platform for constructing advanced NIR-II organic probes. However, their brightness remains suboptimal for practical applications due to intrinsic molecular constraints and environmental quenching effects. Addressing these issues, a high-brightness NIR-II AIE nanoprobe, TT12,8-B NPs, was developed based on the proposed design strategy of increasing molecular alkyl lengths, which simultaneously enhances aggregate hydrophobicity and expands intermolecular packing distance, synergistically improving both molar absorptivity and quantum yield (QY). The optimized TT12,8-B NPs exhibit nearly 4-fold higher brightness than TPE-BBT NPs, one of the highest-QY NIR-II AIE probes reported. Coupled with good photostability and biocompatibility, TT12,8-B NPs enabled high-clarity multiscale vasculature imaging in mice and rabbits, attaining a high signal-to-background ratio exceeding 10 and a tiny vascular resolution of 49 μm in diameter, moving forward in NIR-II fluorescence angiography. Their exceptional angiographic performance further facilitated early tumor detection and discrimination between nascent and established tumors. Beyond the introduction of an excellent NIR-II fluorophore, this work provides an advanced molecular design philosophy.

ABSTRACT Three‐photon (3P) fluorescence imaging (FLI) utilizing excitation wavelengths within the near‐infrared‐III (NIR‐III, 1600–1870 nm) window has emerged as a transformative modality for intravital imaging, owing to its combined advantages of excellent spatiotemporal resolution and remarkable tissue penetration. High‐performance fluorescent probes are the cornerstone of high‐quality NIR‐III 3P FLI. However, the construction of such probes is often hindered by inherent trade‐offs in molecular design principles, posing significant challenges for their performance optimization and practical application. Here, we propose a straightforward and effective strategy based on π‐bridge manipulation to reconcile those competing molecular design parameters and substantially enhance 3P fluorescence properties. Leveraging this approach, a robust AIE‐active small molecule, named TSSID, was developed, which exhibits bright NIR‐I (700–950 nm) emission under 1665 nm NIR‐III 3P excitation when formulated into nanoparticles (NPs). Remarkably, upon retro‐orbital injection into mice following craniotomy, TSSID NPs achieved the best performance in deep‐brain angiography among all reported organic 3P materials in terms of vascular imaging depth, signal‐to‐background ratio, spatial resolution, and hemodynamic imaging depth. Additionally, TSSID NPs demonstrated outstanding biocompatibility through systematic biosafety evaluations. This study provides an excellent imaging agent and useful molecular design philosophy, facilitating the development of advanced organic 3P FLI probes.

Photo-immunotherapy is one of the most promising cancer treatment strategies. As immunotherapeutic agents, immune checkpoint blockade antibodies against programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1) exhibit substantial potential, but have to face non-specific distribution and the subsequent immune-related adverse events. Meanwhile, high-performance phototheranostic agents concurrently possessing multiple phototheranostic modalities and high light-harvesting capacity are really attractive and highly desired as touching phototheranostic modules. Herein, a win-win strategy that integrates phototheranostic molecule design and targeted immunotherapeutic module preparation is developed to construct high-powered photo-immunotherapy systems. Specifically, the phototheranostic agent (AOTTIT) displaying typical aggregation-induced fluorescence extending to the second near-infrared II window, as well as outstanding reactive oxygen species and heat production capacity is first obtained via ingenious design. Notably, AOTTIT exhibits a record high molar extinction coefficient among the reported organic multimodal phototheranostic molecules. Meanwhile, PD-1 genetically engineered cancer cell membrane-derived nanovesicles (PD-1/CMNVs) are prepared as both nanocarriers and immunotherapeutic agents to camouflage AOTTIT nanoparticles, yielding a multifunctional photo-immunotherapeutic agent (CMNPs/PD-1) with tumor-specific active and homologous targeting ability. The distinct suppression of primary and metastatic lung tumors after only once treatment to the primary tumor substantiated the synergistically strengthened photo-immunotherapeutic efficiency of this win-win strategy.

This study aimed to create a new recombinant virus by modifying the EV-A71 capsid protein, serving as a useful tool and model for studying human Enteroviruses. We developed a new screening method using EV-A71 pseudovirus particles to systematically identify suitable insertion sites and tag types in the VP1 capsid protein. The pseudovirus’s infectivity and replication can be assessed by measuring postinfection luciferase signals. We reported that the site after the 100th amino acid within the VP1 BC loop of EV-A71 is particularly permissive for the insertion of various tags. Notably, the introduction of S and V5 tags at this position had minimal effect on the fitness of the tagged pseudovirus. Furthermore, recombinant infectious EV-A71 strains tagged with S and V5 epitopes were successfully rescued, and the stability of these tags was verified. Computational analysis suggested that viable insertions should be compatible with capsid assembly and receptor binding, whereas non-viable insertions could potentially disrupt the capsid’s binding with heparan sulfate. We expect the tagged recombinant EV-A71 to be a useful tool for studying the various stages of the enterovirus life cycle and for virus purification, immunoprecipitation, and research in immunology and vaccine development. Furthermore, this study serves as a proof of principle and may help develop similar tags in enteroviruses, for which there are fewer available tools.