Ke Xu

We report a generalizable method for single-molecule localization super-resolution microscopy in living cells. Live-cell PAINT (points accumulation for imaging in nanoscale topography) and single-molecule diffusivity mapping (SM d M) of intracellular protein targets are achieved by pairing exceptionally fluorogenic bis-trifluoromethyl rhodamine (BF) dyes with reversible HaloTag ligands and widely available HaloTag fusion proteins. This far-red small-molecule update to protein-based PAINT is readily incorporated into existing super-resolution microscopy workflows: wash-free staining and imaging are achieved for cell lines and primary neuron cultures for diverse protein targets. Pairing with photoconvertible fluorescent proteins further enables simultaneous two-color live-cell super-resolution microscopy and SM d M.

We report a framework for quantifying the in situ reaction kinetics of bio­(macro)­molecules in the solution phase through single-molecule displacement statistics. Using single-molecule displacement/diffusivity mapping (SMdM), we measure the transient (0.6 ms) displacements of freely diffusing molecules in the wide field at ultrahigh throughput. Fitting the time-dependent displacement distributions of many molecules to a two-component diffusion model quantifies the reactant and product fractions based on molecular-weight differences, thereby enabling kinetic analysis of reaction progress at ∼1 s temporal resolution. Working with ∼100 pM fluorescently tagged reactant while varying the concentration of a second, unlabeled reactant over wide ranges, we determine second-order reaction rate constants for strain-promoted azide–alkyne cycloaddition and N-hydroxysuccinimide ester aminolysis as well as first-order hydrolysis side-reaction rate constants for the latter. For antibody binding kinetics, we quantify association and dissociation rate constants for a monoclonal antibody and unveil the gradual formation of cross-linked complexes/aggregates for a polyclonal antibody. Working with microliter-scale samples, our single-molecule-displacement-based approach provides intuitive readouts linked to the molecular size and is applicable to diverse reaction types and conditions.

Electron transfer (ET) reactions underpin energy conversion and chemical transformations in both biological ^1,2 and abiological ^3–5 systems. The efficiency of any ET process relies on achieving a desired ET rate within an optimal driving force range. Marcus theory ^6,7 provides a microscopic framework for understanding the activation free energy—and therefore the rate—of ET in terms of a key parameter: the reorganization energy. For electrified solid–liquid interfaces, it has long been conventionally understood that only factors in the electrolyte phase are responsible for determining the reorganization energy and that the electronic density of states (DOS) of the electrode only serves to dictate the number of thermally accessible channels for ET ^5,8–12 . Here we show instead that the electrode DOS plays a central role in governing the reorganization energy, far outweighing its conventionally assumed role. Using atomically layered heterostructures, we tune the DOS of graphene and measure outer-sphere ET kinetics. We find the ensuing variation in ET rate arises from strong modulation in a reorganization energy associated with image potential localization in the electrode. Here we redefine the traditional paradigm of heterogeneous ET kinetics, revealing a deeper role of the electrode electronic structure in interfacial reactivity.

Single‐walled carbon nanotubes (SWCNTs) show promise for probing molecular interactions at single‐molecule resolution, yet generating SWCNT populations bearing a single defined functional tag remains challenging because surface functionalization is inherently stochastic. Here, we present a batch‐scale strategy to produce predominantly singly tagged SWCNTs by leveraging the stochastic adsorption of single‐stranded DNA (ssDNA). Specifically, SWCNTs are dispersed using a mixture of unmodified ssDNA (um‐ssDNA) and a minor fraction of modified ssDNA (m‐ssDNA) carrying an affinity handle. We developed a probabilistic ssDNA–SWCNT binding model that predicts the distribution of m‐ssDNA per nanotube as a function of the input minor‐strand fraction p = m‐ssDNA/total ssDNA, enabling selection of conditions that maximize single‐tag purity. Using magnetic‐bead capture via a biotin affinity interaction and subsequent release, we isolate SWCNTs with 97.6% predicted single‐tag purity at 2% recovery. Single‐molecule fluorescence imaging further supports predominantly single‐label occupancy under the model‐selected conditions. Thus, this approach provides a general route to SWCNTs bearing a single molecular handle for downstream conjugation and assembly, supporting diverse future applications in SWCNT‐based nanotechnologies.

Single‐walled carbon nanotubes (SWCNTs) show promise for probing molecular interactions at single‐molecule resolution, yet generating SWCNT populations bearing a single defined functional tag remains challenging because surface functionalization is inherently stochastic. Here, we present a batch‐scale strategy to produce predominantly singly tagged SWCNTs by leveraging the stochastic adsorption of single‐stranded DNA (ssDNA). Specifically, SWCNTs are dispersed using a mixture of unmodified ssDNA (um‐ssDNA) and a minor fraction of modified ssDNA (m‐ssDNA) carrying an affinity handle. We developed a probabilistic ssDNA–SWCNT binding model that predicts the distribution of m‐ssDNA per nanotube as a function of the input minor‐strand fraction p = m‐ssDNA/total ssDNA, enabling selection of conditions that maximize single‐tag purity. Using magnetic‐bead capture via a biotin affinity interaction and subsequent release, we isolate SWCNTs with 97.6% predicted single‐tag purity at 2% recovery. Single‐molecule fluorescence imaging further supports predominantly single‐label occupancy under the model‐selected conditions. Thus, this approach provides a general route to SWCNTs bearing a single molecular handle for downstream conjugation and assembly, supporting diverse future applications in SWCNT‐based nanotechnologies.

Clathrin-mediated endocytosis (CME) is an essential cellular process that needs to operate efficiently across a wide range of conditions. Internalization of the endocytic site involves forces generated by membrane-bound proteins and Arp2/3-mediated branched actin filament assembly to bend the plasma membrane from flat to omega-shaped. In mammalian CME, the requirement for a branched actin filament network varies depending on cell type and differences in membrane tension. However, how the actin network adapts to changes in load in order to ensure robustness of this process over a range of membrane tensions is not understood. Here, we combine live-cell imaging and superresolution microscopy of genome-edited human induced pluripotent stem cells to investigate the role of the mammalian class-I myosin, Myosin1E (Myo1E), in load adaptation. Under normal conditions, sites that recruit Myo1E are rare and exhibit slow CME dynamics. However, as membrane tension increases and CME dynamics are slowed globally, Myo1E is recruited to more sites, likely to increase actin assembly and motor activity, resulting in increased force generation to rescue stalled sites and promote internalization. Loss of Myo1E results in increased Arp2/3 complex lifetime at CME sites under normal conditions, and at high membrane tension, these sites fail to recruit as many Arp2/3 molecules. We propose that Myo1E is recruited to CME sites that have stalled due to increased membrane tension, where it helps build a more effective branched actin network by generating force through motor activity and recruiting additional Arp2/3 complexes to rescue stalled sites.

Stacked (phospho)lipid bilayers are ubiquitous and of both fundamental and applicational importance. We demonstrate fast, facile label-free microscopic visualization and quantification of glass-supported stacked lipid bilayers (SSLBs) in aqueous environments through interference reflection microscopy (IRM). Quantitative analysis of three-wavelength IRM shows good agreement with theory, achieving ∼10% signal contrast for each ∼5 nm-thick stacked bilayer and definite designation of local bilayer numbers. Harnessing this capability, we show that SSLBs, with ordered domains tens of micrometers in size, are readily formed under diverse conditions of spin-coating, evaporation from organic solutions, and deposition from liposome dispersions. Moreover, we find that SSLBs are stable under physiological ionic strengths but are destabilized in low-salt conditions and prone to delamination in pure water, under which condition local peeling and wrinkling occur for the stacked bilayers but not for the substrate-adhered basal bilayer. Consequently, washing away the stacked bilayers with water leaves behind a uniform single supported lipid bilayer (SLB). Single-molecule displacement/diffusivity mapping shows that this single bilayer exhibits diffusivity indistinguishable from conventional SLBs. In contrast, stacked bilayers exhibit ∼60% higher diffusivity, indicative of a more native environment than SLBs due to decoupling from substrate interactions. Together, our results reveal the ubiquity, microscopic structure, and properties of SSLBs, providing fundamental insights and pointing to potential applications.

Wide-field single-molecule tracking is often limited by the ~10 ms camera frame time. We introduce SpeedyTrack, which directly enables microsecond wide-field single-molecule tracking/imaging on common setups. Harnessing the native sub-microsecond vertical charge shifting capability of EM-CCDs, SpeedyTrack staggers wide-field single-molecule images along the CCD chip at ~10-row spacings between consecutive timepoints, effectively projecting the time domain to the spatial domain. Wide-field tracking is achieved for molecules diffusing at up to 1000 µm ² /s at 50 µs temporal resolutions for >30 timepoints. Concurrent Förster resonance energy transfer measurements further elucidate molecular states. By implementing temporally patterned vertical shifting, VS-SpeedyTrack next deconvolves the spatial and temporal information to map trajectories at the super-resolution level, resolving the diffusion mode of a fluorescent protein in live cells with nanoscale resolution. Without modifications to existing optics or electronics, SpeedyTrack provides a facile solution to the microsecond tracking of single molecules and their super-resolution mapping in the wide field.

Clathrin-mediated endocytosis (CME) is an essential cellular process that needs to operate efficiently across a wide range of conditions. Internalization of the endocytic site involves forces generated by membrane-bound proteins and Arp2/3-mediated branched actin filament assembly to bend the plasma membrane (PM) from flat to omega-shaped. In mammalian CME, the requirement for a branched actin filament network varies depending on cell type and differences in membrane tension. However, how the actin network adapts to changes in load in order to ensure robustness of this process over a range of membrane tensions is not understood. Here, we combine live-cell imaging and super-resolution microscopy of genome-edited human induced pluripotent stem cells (hiPSCs) to investigate the role of the mammalian class-I myosin, Myosin1E (Myo1E), in load adaptation. Under normal conditions, sites that recruit Myo1E are rare and exhibit slow CME dynamics. However, as membrane tension increases and CME dynamics are slowed globally, Myo1E is recruited to more sites, likely to assemble more branched actin, resulting in increased force generation to rescue stalled sites and promote internalization. Loss of Myo1E results in increased Arp2/3 complex lifetime at CME sites under normal conditions, and at high membrane tension, these sites fail to recruit as many Arp2/3 molecules. We propose that Myo1E is recruited to CME sites that have stalled due to increased membrane tension, where it helps build a more effective branched actin network by generating force through motor activity and recruiting additional Arp2/3 complexes to rescue stalled sites. Significance: For mammalian cells to internalize extracellular cargo via clathrin-mediated endocytosis (CME), specific regions of the plasma membrane (PM) must bend from flat to inwardly curved, a process that requires force-generating proteins. One key component in generating this force during CME is the branched actin network, in which actin filaments polymerize against the plasma membrane. When PM tension increases, more force is required to generate curvature, prompting the assembly of actin and actin associated proteins to aid the process. We demonstrate that the class-I myosin motor, Myosin1E (Myo1E), becomes increasingly crucial as membrane tension rises, presumably to build a more effective branched actin network to facilitate internalization of slowed sites.

The molecular mechanism of general anesthesia remains a mystery. While many small molecules, ranging from xenon to diethyl ether, act as general anesthetics, few similarities exist in their chemical structures or properties. Utilizing single-molecule displacement/diffusivity mapping (SMdM), a diffusion-quantifying single-molecule and super-resolution microscopy tool, we unveil that at clinical concentrations, general anesthetics rapidly and reversibly enhance the lateral diffusivity of both model lipid bilayers and live-cell plasma membranes in a dose-dependent fashion based on the anesthetic potency. With in situ fluorescence microscopy, we next show that the partitioning of anesthetics into the lipid bilayer causes fast dilation and area expansion. Employing a liposome-based fluorescence quenching assay, we further unveil enhanced lipid-bilayer permeability to the chloride ion (Cl^-) in an anesthetic-concentration-dependent fashion. Together, our results indicate that the reversible insertion of anesthetic molecules into the lipid bilayer enhances the diffusivity and permeability of the lipid membrane, thus compromising neural functions.