Metal-organic frameworks with emissive ligands offer tunable photophysical properties that are sensitive to their coordination environment and structural configuration. In this study, we demonstrate that the fluorescence of UiO-66-NH2 can be modulated through photo-oxidation-induced quenching and subsequent solvent-mediated recovery. Upon visible-light irradiation, reactive oxygen species induce two distinct oxidation pathways: irreversible oxidation of the ligand and reversible oxidation of the metal-oxo cluster. These redox events lead to partial disruption of the coordination environment and result in fluorescence loss. Reduction using mild alcohols selectively removes adsorbed oxidative species from Zr6O4(OH)4, leading to partial recovery of fluorescence and porosity. The extent of fluorescence restoration correlates with both the reducing strength and molecular size of the alcohol, reflecting its ability to penetrate internal pores and access to redox-active sites. Notably, some alcohol-treated samples exhibited nitrogen uptake beyond that of pristine UiO-66-NH2, suggesting redux-induced structural reorganization and defect-assisted pore expansion. These results establish a structure-function relationship in MOFs governed by localized redox chemistry, providing a platform for designing reconfigurable optical materials with switchable photophysical and porous characteristics.

Recent research has explored the anticancer properties of immune-cell-derived small extracellular vesicles (sEVs), but many challenges, like the need for improved targeting, remain. To address these challenges, we engineered T-cell-derived sEVs with antitransferrin receptor 1 (TfR1) antibodies (T-EVs). This modification enhanced the delivery of sEV to six types of cancer cells, as confirmed by flow cytometry, immunocytochemistry, live cell imaging, and blocking experiments in vitro. The T-EVs also reduced PD-L1 and Rab27a levels, decreased sEV production from breast cancer cells, and increased susceptibility to CD8+ T-cell-mediated cytotoxicity. Systemically administered T-EVs efficiently targeted breast, lung, and skin tumors in mouse models. Notably, T-EVs significantly inhibited tumor growth without systemic toxicity. Additionally, T-EVs reduced PD-L1 and Rab27a levels in cancer cells while enhancing the CD8+ T-cell cytotoxicity and proliferation. Overall, this study highlights the anticancer effects of T-EVs against multiple cancer types, underscoring their potential in developing targeted cancer therapies.

Although gold is regarded as inert, its redox cycling produces reactive oxygen species that compromise cellular integrity. Alternatively, nickel stabilises upon oxidation, limiting charge transfer and oxidative stress. We show that Au-based substrates disrupt organelles, whereas Ni-based substrates preserve endoplasmic reticulum tubules, compelling the reassessment of material biocompatibility in composite systems.

Current chemical strategies for modifying the surface of extracellular vesicles (sEVs) often struggle to balance efficient functionalization with preserving structural integrity. Here, we present a modular approach for the surface modification of sEVs using a chimeric adaptor protein (CAP). The CAP was designed with three key features: a SNAP-tag for stable and modular binding, long and rigid linker to enhance spatial accessibility and conjugation efficiency, and the N-terminal sorting domain derived from syntenin to improve CAP expression on the sEV. We established a postsynthetic method to introduce diverse functional molecules onto sEVs, creating a versatile system termed "sEV-X" (where X represents an organic molecule, protein, or nanoparticle). Quantitative analyses at the single-molecule level revealed a linear relationship between CAP expression and the number of conjugated functional molecules, underscoring the importance of steric hindrance mitigation in sEV surface engineering. Moreover, antibody-conjugated sEVs as drug carriers, demonstrated significant tumor-specific delivery and therapeutic efficacy in a tumor-bearing mouse model, underscoring the potential of CAP-expressing sEVs as a customizable therapeutic vesicle. Overall, the CAP technology may serve as a universal platform for advancing the development of sEV-based therapeutics.

Electric field (e-field)-based therapies like tumor-treating fields (TTFields) are promising non-invasive cancer treatments, but their clinical utility is limited by shallow tissue penetration, low spatial specificity, and potential thermal side effects. This study presents surface-engineered tetragonal phase barium titanate nanoparticles (tBTO NPs) that locally amplify weak e-fields to improve therapy. Stable dispersion and targeted delivery of tBTO NPs in biological environments, without detectable cytotoxicity, are achieved through precise surface chemical modifications. Comparative experiments underscore the critical role of ferroelectricity: tBTO NPs exhibit superior microtubule disruption and cell growth inhibition than non-ferroelectric Au or low-ferroelectric cubic phase BTO NPs. Super-resolution microscopy and single-cell tracking quantitatively demonstrate how amplified e-fields perturb cellular behaviors, including migration, proliferation, and morphology. Overall, tBTO NPs may address TTField limitations and advance nanomaterial-based bioelectronic cancer therapies, broadening the use of electromagnetic technologies in precision medicine.

Investigation of the fundamental microscopic processes occurring in organic reactions is essential for optimising both organocatalysts and synthetic strategies. In this study, single-molecule fluorescence microscopy was employed to study the Diels–Alder reaction catalysed by a first-generation MacMillan catalyst, providing direct insights into its kinetic dynamics. This reaction proceeds via a series of reversible processes under equilibrium conditions ( S ⇄ IM 1 ⇄ IM 2 → P , IM 1 and IM 2 : N,O-acetal and iminium ion intermediates, respectively). The individual reaction trajectories of single molecules were directly observed in real-time, and the kinetic transitions between the different states were quantitatively analysed using a hidden Markov model, thereby enabling precise determination of the kinetic rate constants and transition probabilities at the single-molecule level. In particular, the unique structural features of the MacMillan catalyst were probed to reveal how specific interactions stabilise the reaction intermediates and influence their kinetic behaviours. These findings highlight the importance of single-molecule fluorescence microscopy in understanding the fundamental mechanisms of organic reactions and guiding the rational design of more effective catalysts.

Epidermal growth factor receptor (EGFR) dimerization plays a pivotal role in cellular signaling, influencing proliferation and disease progression, particularly in cancer. Despite extensive studies, the quantitative relationship between EGFR expression levels and dimerization efficiency remains incompletely understood. In this study, we investigated EGFR dimerization kinetics using ensemble-level biochemical assays and single-molecule tracking (SMT) in living cells. Our findings revealed noncanonical negative cooperative dimerization, where the monomer-to-dimer transition rate decreased as EGFR expression increased, challenging the assumptions of a simplistic reaction model. Furthermore, we identified a dimer-specific degradation pathway highlighting the open-system nature of the plasma membrane environment. These findings establish a quantitative framework for understanding EGFR dimerization dynamics, offering insights into the complex regulatory principles governing membrane protein interactions. This model not only improves our understanding of EGFR-mediated signaling but also suggests broader applicability for the therapeutic targeting of membrane protein systems.

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disorder with a complex pathogenesis that requires combination therapies rather than monotherapies. Extracellular vesicles (EVs) exhibit inherently efficient delivery to the liver and can be engineered to carry various therapeutic substances, making them promising agents. In this study, EVs were engineered to display fibroblast growth factor 21 (FGF21) on their surface and encapsulate miR-223 (223/F-EVs), aiming to improve steatosis and alleviate inflammation and fibrosis, respectively. Introducing the 223/F-EVs into human liver cell lines significantly reduced both basal and induced levels of lipid storage, inflammation, and fibrosis markers. Furthermore, using an FGF21-blocking antibody or miR-223 inhibitor effectively diminished the efficacy of the 223/F-EVs, confirming the essential roles of FGF21 and miR-223 in these processes. In a Choline-Deficient, l-Amino acid-defined, High-Fat Diet (CDAHFD)-fed mouse model, intravenously administered 223/F-EVs demonstrated liver-preferential delivery and a marked reduction in the MASH phenotype without compromising bone density, unlike conventional FGF21 treatment. Collectively, 223/F-EVs convey FGF21 and miR-223 exclusively to the liver, offering strategic advantages by mitigating MASH progression via multiple pathways. This study lays a solid foundation for further investigation of engineered EVs as a transformative therapeutic approach for treating MASH.

The calcium-dependent phospholipid scramblase TMEM16E mediates ion transport and lipid translocation across the plasma membrane. TMEM16E also contributes to protection of membrane structure by facilitating cellular repair signaling. Our research reveals that TMEM16E activation promotes macropinocytosis, essential for maintaining plasma membrane integrity. This scramblase externalizes phosphatidylserine, typically linked to resting growth factor receptors. We demonstrate that TMEM16E can interact with and signal through growth factor receptors, including epidermal growth factor receptor, even without ligands. This interaction stimulates downstream phosphoinositide 3-kinase and facilitates macropinocytosis and internalization of annexin V bound to the membrane, a process sensitive to amiloride inhibition. Although TMEM16E is internalized during this process, it returns to the plasma membrane. TMEM16E- driven macropinocytosis is proposed to restore membrane integrity after perturbation, potentially explaining pathologies in conditions like muscular dystrophies, where TMEM16E functionality is compromised, highlighting its critical role in muscle cell survival. TMEM16E is a calcium-dependent scramblase that mediates ion transport and lipid translocation, contributing to membrane protection. This study shows TMEM16E promotes macropinocytosis, crucial for maintaining plasma membrane integrity.

Death receptor (DR) is a unique transmembrane receptor mediating extrinsic factor-induced programmed cell death pathways. For most DRs, the regulatory mechanism has been revealed to involve agonistic death ligand (e.g., TRAIL, TNF-alpha)-driven receptor clustering and activation, offering an in-depth understanding of the mechanistic basis for cell death regulation and therapeutic strategies. However, only death receptor 6 (DR6) remains orphan, and its molecular mechanism is poorly understood. Here, we identified retinol binding protein 4 (RBP4), a major vitamin-A carrier protein, as an antagonistic ligand of DR6. Single-molecule photobleaching and single-receptor tracking analyses revealed that RBP4 drives DR6 dimerization on the plasma membrane to prevent DR6 prodegenerative hetero-interactions and consequent neurodegenerative processes. DR6 homodimerization predominantly depends on an extracellular intrinsically disordered region, allowing the agonistic propensity of increasing Ca2+ levels. Collectively, our study establishes the distinct molecular mechanism of DR6 and highlights the non-canonical role of RBP4 as a cell death regulator. ### Competing Interest Statement The authors have declared no competing interest.

We have rationally designed a one-dimensional coordination polymer (1D CP), termed 1D-DGIST-18, that exhibits intrinsic structural flexibility. This 1D CP enables its expansion into a three-dimensional network through supramolecular interactions involving coordinated solvents and/or ligands. The strategic selection of solvents for solvent exchange, prior to drying, significantly influences the structures of 1D-DGIST-18 by removing certain coordinating solvents and modulating π-π stacking. Consequently, a hierarchical porosity emerges, ranging from micro- to meso- to macroporous structures, which is attributed to its inherent structural dynamics. Additionally, the formation of excimers endows 1D-DGIST-18, when immersed in acetone, with 'turn-on' fluorescence, as evidenced by fluorescence decay profiles. These structural transitions within 1D-DGIST-18 are further elucidated using single-crystal X-ray diffractometry. The insights from this study provide a foundation for the design of materials with structural dynamics and tunable properties.

The material transport system, facilitated by motor proteins, plays a vital role in maintaining a non-equilibrium cellular state. However, understanding the temporal coordination of motor protein activity requires an advanced imaging technique capable of measuring 3D angular displacement in real-time. In this study, a Fourier transform-based plasmonic dark-field microscope has been developed using anisotropic nanoparticles, enabling the prolonged and simultaneous observation of endosomal lateral and rotational motion. A sequence of discontinuous 3D angular displacements has been observed during the pause and run phases of transport. Notably, a serially correlated temporal pattern in the intermittent rotational events has been demonstrated during the tug-of-war mechanism, indicating Markovian switching between the exploitational and explorational modes of motor protein exchange prior to resuming movement. Alterations in transition frequency and the exploitation-to-exploration ratio upon dynein inhibitor treatment highlight the relationship between disrupted motor coordination and reduced endosomal transport efficiency. Collectively, these results suggest the importance of orchestrated temporal motor protein patterns for efficient cellular transport.

Understanding the spatial organization of membrane proteins is crucial for unraveling key principles in cell biology. The reaction-diffusion model is commonly used to understand biochemical patterning; however, applying reaction-diffusion models to subcellular phenomena is challenging because of the difficulty in measuring protein diffusivity and interaction kinetics in the living cell. In this work, we investigated the self-organization of the plasmalemma vesicle-associated protein (PLVAP), which creates regular arrangements of fenestrated ultrastructures, using single-molecule tracking. We demonstrated that the spatial organization of the ultrastructures is associated with a decrease in the association rate by actin destabilization. We also constructed a reaction-diffusion model that accurately generates a hexagonal array with the same 130 nm spacing as the actual scale and informs the stoichiometry of the ultrastructure, which can be discerned only through electron microscopy. Through this study, we integrated single-molecule experiments and reaction-diffusion modeling to surpass the limitations of static imaging tools and proposed emergent properties of the PLVAP ultrastructure.

The biological process of aging is thought to result in part from accumulation of senescent cells in organs. However, the present study identified a subset of fibroblasts and smooth muscle cells which are the major constituents of organ stroma neither proliferative nor senescent in tissues of the elderly, which we termed "mid-old status" cells. Upregulation of pro-inflammatory genes (IL1B and SAA1) and downregulation of anti-inflammatory genes (SLIT2 and CXCL12) were detected in mid-old cells. In the stroma, SAA1 promotes development of the inflammatory microenvironment via upregulation of MMP9, which decreases the stability of epithelial cells present on the basement membrane, decreasing epithelial cell function. Remarkably, the microenvironmental change and the functional decline of mid-old cells could be reversed by a young cell-originated protein, SLIT2. Our data identify functional reversion of mid-old cells as a potential method to prevent or ameliorate aspects of aging-related tissue dysfunction.

Optical nanoscopy, also known as super-resolution optical microscopy, has provided scientists with the means to surpass the diffraction limit of light microscopy and attain new insights into nanoscopic structures and processes that were previously inaccessible. In recent decades, numerous studies have endeavored to enhance super-resolution microscopy in terms of its spatial (lateral) resolution, axial resolution, and temporal resolution. In this review, we discuss recent efforts to push the resolution limit of stimulated emission depletion (STED) optical nanoscopy across multiple dimensions, including lateral resolution, axial resolution, temporal resolution, and labeling precision. We introduce promising techniques and methodologies building on the STED concept that have emerged in the field, such as MINSTED, isotropic STED, and event-triggered STED, and evaluate their respective strengths and limitations. Moreover, we discuss trade-off relationships that exist in far-field optical microscopy and how they come about in STED optical nanoscopy. By examining the latest developments addressing these aspects, we aim to provide an updated overview of the current state of STED nanoscopy and its potential for future research.

Recent techniques for direct observation of single molecules or nanoparticles provide methodologies for imaging the activation sites of heterogeneous catalysts (spatially resolved) and observing intermediates that are not visible in the ensemble average (temporally resolved). Accordingly, the primary challenge for related experiments is obtaining sufficient spatial and temporal resolutions for microscopic observation of the chemical reaction of interest. This review discusses recent advances in fluorescence-for example, total internal reflection fluorescence (TIRF)-and dark-field microscopy-for example, imaging plasmonic probes-used for observing organic, inorganic, and biological reactions. The following key factors for microscopic observation of chemical reactions are discussed: (1) design of the chemical reaction and probe, (2) selection of microscope based on reaction's temporal information, and (3) use of machine learning algorithms to analyze the sequence imaging data. This review summarizes experimental techniques and detailed examples of reactions at the single molecule and nanoparticle level. Furthermore, it discusses avenues of development. These observations can guide the development of new and systematic methodological approaches for investigating important unsolved problems in chemistry.

In live cells, the plasma membrane is composed of lipid domains separated by hundreds of nanometers in dynamic equilibrium. Lipid phase separation regulates the trafficking and spatiotemporal organization of membrane molecules that promote signal transduction. However, visualizing domains with adequate spatiotemporal accuracy remains challenging because of their subdiffraction limit size and highly dynamic properties. Here, we present a single lipid-molecular motion analysis pipeline (lipid-MAP) for analyzing the phase heterogeneity of lipid membranes by detecting the instantaneous velocity change of a single lipid molecule using the excellent optical properties of nanoparticles, high spatial localization accuracy of single-molecule localization microscopy, and separation capability of the diffusion state of the hidden Markov model algorithm. Using lipid-MAP, individual lipid molecules were found to be in dynamic equilibrium between two statistically distinguishable phases, leading to the formation of small (similar to 170 nm), viscous (2.5x more viscous than surrounding areas), and transient domains in live cells. Moreover, our findings provide an understanding of how membrane compositional changes, i.e., cholesterol and phospholipids, affect domain formation. This imaging method can contribute to an improved understanding of spatiotemporal-controlled membrane dynamics at the molecular level.

Live video recording of intracellular material transport is a promising means of deciphering the fascinating underlying mechanisms driving life at the molecular level. Such technology holds the key to realizing real-time observation at appropriate resolutions in three-dimensional (3D) space within living cells. Here, we report an optical microscopic method for probing endosomal dynamics with proper spatiotemporal resolution within 3D space in live cells: plasmonic dark-field STORM (pdf-STORM). We first confirmed that pdf-STORM has a spatial resolution comparable to that of scanning electron microscopy. Additionally, by observing two optical probes within a single organelle, we were able to track rotational movements and demonstrate the feasibility of using pdf-STORM to observe the angular displacements of an endosome during a "tug-of-war" over an extended period. Finally, we show various biophysical parameters of the hitherto unelucidated dynamics of endosomes-angular displacement is discontinuous and y-axis movement predominates and follows a long-tail distribution.

Intracellular cargos are transported in the form of endosomes by motor proteins along a microtubule network. The mechanical competition between different motors, i.e. kinesin and dynein, called an endosomal “tug-of-war'', regulates not only metabolite movements but also direction of transport associated with in signaling transduction pathways, which is also spatially and temporally regulated. However, how their dynamic movements, i.e. pause, run, and turn, and their kinetic parameters, is unclear due to technical challenge for observation.

In live cell, plasma membrane consisted of separated phases which is in dynamic equilibrium. The phase separation regulate behaviors of the biomolecules as well as downstream signaling. Due to the sub-diffraction limited size (< 200 nm) of the viscous nanodomain (i.e., lipid raft), the molecular time trace at a high space-time accuracy is important for visualization of multiphase nature of the membrane. We analyze individual molecular motion with high localization accuracy by a single molecule localization microscopy (SMLM) and the hidden Markov’s model (HMM).