Angstrom Precision Research Articles

Direct optical measurement of intramolecular distances with angstrom precision.

Optical investigations of nanometer distances between proteins, their subunits, or other biomolecules have been the exclusive prerogative of Förster resonance energy transfer (FRET) microscopy for decades. In this work, we show that MINFLUX fluorescence nanoscopy measures intramolecular distances down to 1 nanometer-and in planar projections down to 1 angstrom-directly, linearly, and with angstrom precision. Our method was validated by quantifying well-characterized 1- to 10-nanometer distances in polypeptides and proteins. Moreover, we visualized the orientations of immunoglobulin subunits, applied the method in human cells, and revealed specific configurations of a histidine kinase PAS domain dimer. Our results open the door for examining proximities and interactions by direct position measurements at the intramacromolecular scale.

Two-Dimensional Sulfonate-Functionalized Metal-Organic Framework Membranes for Efficient Lithium-Ion Sieving.

Two-dimensional (2D) membranes have shown promising potential for ion-selective separation but often suffer from the trade-off between permeability and selectivity. Herein, we report an ultrathin 2D sulfonate-functionalized metal-organic framework (MOF) membrane for efficient lithium-ion sieving. The narrow pores with angstrom precision in the MOF assist hydrated ions to partially remove the hydration shell, according to different hydration energies. The abundant sulfonate groups in the MOF channels serve as hopping sites for fast lithium-ion transport, contributing to a high Li-ion permeability. Then, the difference in affinity of the Li+, Na+, K+, and Mg2+ ions to the terminal sulfonate groups further enhances the Li-ion selectivity. The reported ultrathin MOF membrane overcomes the trade-off between permeability and selectivity and opens up a new avenue for highly permselective membranes.

Angstrom-precise fabrication of surface nanoscale axial photonics (SNAP) microresonators with a flame

We demonstrate the fabrication of surface nanoscale axial photonics bottle microresonators with angstrom precision using a flame. We observe strongly unscalable behavior of the whispering gallery mode cutoff wavelengths with different radial quantum numbers along the fibre length.

Probing the polar-nonpolar oxide interfaces using resonant x-ray standing wave techniques

Transition metal (TM) oxide heterostructure superlattices have attracted great attention in research communities because of their emergent interfacial phenomena that do not exist in the bulk form. In order to understand the mechanisms that cause these phenomena, it is important to use depth-resolved spectroscopies to study the electronic structure across the buried oxide interfaces. In this review, we focus on the recent applications of standing wave (SW) photoemission (SW-XPS) and resonant inelastic x-ray scattering (SW-RIXS) spectroscopy to study the depth profiles of an electronic structure or carriers around the polar-nonpolar oxide interfaces. Using the incident photon energies near the TM x-ray absorption resonance, the created SW excitation can enhance the spectral response and certain electronic transitions, providing important insight into the interfacial electronic structure in the energy and real space regimes. Following the background introductions, we describe two SW experiments and demonstrate that the combination of SW-XPS and SW-RIXS has the potential to obtain the depth distribution of electronic/orbital states around the buried interfaces with Angstrom precision.

In-situ control of on-chip angstrom gaps, atomic switches, and molecular junctions by light irradiation

Pairs of electrodes with nanometer gap, termed as nano-gapped electrodes, are fundamental building blocks for the fabrication of nanometer-sized devices and are essential for the examination of molecular properties and extreme nano-optics. Although modern fabrication techniques make it feasible to fabricate nanometer gaps, it is still a formidable challenge to fabricate adjustable gaps arrays with angstrom precision. Here, we demonstrate that in-situ adjustable nanogaps (arrays) with sub-angstrom precision can be fabricated via laser irradiation on the substrate which supports the electrode pairs. We further demonstrate that atomic-level metal contacts can be switched and the direction of the switching can be selectively controlled by the laser irradiation position. By varying the laser power gradually, the nanogap’s size can be continuously changed, providing a reliable break junction technique to address the properties of single-molecule junctions. The small spatial focus size of the laser beam makes it feasible to realize addressable on-chip molecular junction arrays.

Spatiotemporal Resolution of Conformational Changes in Biomolecules by Combining Pulsed Electron-Electron Double Resonance Spectroscopy with Microsecond Freeze-Hyperquenching.

The function of proteins is linked to their conformations that can be resolved with several high-resolution methods. However, only a few methods can provide the temporal order of intermediates and conformational changes, with each having its limitations. Here, we combine pulsed electron-electron double resonance spectroscopy with a microsecond freeze-hyperquenching setup to achieve spatiotemporal resolution in the angstrom range and lower microsecond time scale. We show that the conformational change of the Cα-helix in the cyclic nucleotide-binding domain of the Mesorhizobium loti potassium channel occurs within about 150 μs and can be resolved with angstrom precision. Thus, this approach holds great promise for obtaining 4D landscapes of conformational changes in biomolecules.

Spin Labeling of RNA Using "Click" Chemistry for Coarse-grained Structure Determination via Pulsed Electron-electron Double Resonance Spectroscopy.

Understanding the function of oligonucleotides on a molecular level requires methods for studying their structure, conformational changes, and internal dynamics. Various biophysical methods exist to achieve this, including the whole toolbox of Electron Paramagnetic Resonance (EPR or ESR) spectroscopy. An EPR method widely used in this regard is Pulsed Electron-Electron Double Resonance (PELDOR or DEER), which provides distances in the nanometer range between electron spins in biomolecules with Angstrom precision, without restriction to the size of the biomolecule, and in solution. Since oligonucleotides inherently do not contain unpaired electrons, these have to be introduced in the form of so-called spin labels. Firstly, this protocol describes how nitroxide spin labels can be site-specifically attached to oligonucleotides using "Click" chemistry. The reaction provides little byproducts, high yields, and is conveniently performed in aqueous solution. Secondly, the protocol details how to run the PELDOR experiment, analyze the data, and derive a coarse-grained structure. Here, emphasis is placed on the pitfalls, requirements for a good dataset, and limits of interpretation; thus, the protocol gives the user a guideline for the whole experiment i.e., from spin labeling, via the PELDOR measurement and data analysis, to the final coarse-grained structure. Graphical abstract: Schematic overview of the workflow described in this protocol: First, the spin-labeling of RNA is described, which is performed as a "Click"-reaction between the alkyne-functionalized RNA strand and the azide group of the spin label. Next, step-by-step instructions are given for setting up PELDOR/DEER distance measurements on the labeled RNA, and for data analysis. Finally, guidelines are provided for building a structural model from the previously analyzed data.

Precisely Tunable Ion Sieving with an Al13-Ti3C2Tx Lamellar Membrane by Controlling Interlayer Spacing.

Two-dimensional (2D) membranes exhibit exceptional properties in molecular separation and transport, which reveals their potential use in various applications. However, ion sieving with 2D membranes is severely restrained due to intercalation-induced swelling. Here, we describe how to efficiently stabilize the lamellar architecture using Keggin Al13 polycations as pillars in a Ti3C2Tx membrane. More importantly, interlayer spacing can be easily adjusted with angstrom precision over a wide range (2.7-11.2 Å) to achieve selective and tunable ion sieving. A membrane with narrow d-spacing demonstrated enhanced selectivity for monovalent ions. When applied in a forward osmosis desalination process, this membrane exhibited high NaCl exclusion (99%) with a fast water flux (0.30 L m-2 h-1 bar-1). A membrane with wide d-spacing showed notable selectivity, which was dependent on the cation valence. When it was applied to acid recovery from iron-based industrial wastewater, the membrane showed good H+/Fe2+ selectivity, which makes it a promising substitute for traditional polymeric membranes. Thus, we introduce a possible route to construct 2D membranes with appropriate structures to satisfy different ion-sieving requirements in diverse environment-, resource-, and energy-related applications.

Tunable Thermoelastic Anisotropy in Hybrid Bragg Stacks with Extreme Polymer Confinement

AbstractControlling thermomechanical anisotropy is important for emerging heat management applications such as thermal interface and electronic packaging materials. Whereas many studies report on thermal transport in anisotropic nanocomposite materials, a fundamental understanding of the interplay between mechanical and thermal properties is missing, due to the lack of measurements of direction‐dependent mechanical properties. In this work, exceptionally coherent and transparent hybrid Bragg stacks made of strictly alternating mica‐type nanosheets (synthetic hectorite) and polymer layers (polyvinylpyrrolidone) were fabricated at large scale. Distinct from ordinary nanocomposites, these stacks display long‐range periodicity, which is tunable down to angstrom precision. A large thermal transport anisotropy (up to 38) is consequently observed, with the high in‐plane thermal conductivity (up to 5.7 W m−1 K−1) exhibiting an effective medium behavior. The unique hybrid material combined with advanced characterization techniques allows correlating the full elastic tensors to the direction‐dependent thermal conductivities. We, therefore, provide a first analysis on how the direction‐dependent Young's and shear moduli influence the flow of heat.

Tunable Thermoelastic Anisotropy in Hybrid Bragg Stacks with Extreme Polymer Confinement.

Controlling thermomechanical anisotropy is important for emerging heat management applications such as thermal interface and electronic packaging materials. Whereas many studies report on thermal transport in anisotropic nanocomposite materials, a fundamental understanding of the interplay between mechanical and thermal properties is missing, due to the lack of measurements of direction‐dependent mechanical properties. In this work, exceptionally coherent and transparent hybrid Bragg stacks made of strictly alternating mica‐type nanosheets (synthetic hectorite) and polymer layers (polyvinylpyrrolidone) were fabricated at large scale. Distinct from ordinary nanocomposites, these stacks display long‐range periodicity, which is tunable down to angstrom precision. A large thermal transport anisotropy (up to 38) is consequently observed, with the high in‐plane thermal conductivity (up to 5.7 W m−1 K−1) exhibiting an effective medium behavior. The unique hybrid material combined with advanced characterization techniques allows correlating the full elastic tensors to the direction‐dependent thermal conductivities. We, therefore, provide a first analysis on how the direction‐dependent Young's and shear moduli influence the flow of heat.

In-Plane Distribution of Water inside Nafion Thin Film Analyzed By Neutron Reflectivity at Temperature of 80 °C and Relative Humidity of 30-80%

Understanding the interfaces in an MEA of a PEFC, or the reaction fields, has become very important, and their structures and properties are intensively studied. Bulk and interfacial structures of polymer electrolyte membranes and binders, as well as the distribution of water inside, are important for designing new ion-conductive ionomers for polymer electrolyte fuel cells. Neutron reflectivity (NR) is a powerful method to observe structures of electrolyte polymers on flat substrates with an Angstrom precision in the in-plane direction. NR measurements were carried out on model Nafion films with a thickness of approximately 100 nm formed on a native SiO2 surface and on a Pt layer on Si(100) for understanding the in-plane Nafion and water densities.1) Under N2 humidified with H2O, the temperature was set at 80 ˚C and the relative humidity between 30% and 80% during the NR measurements, simulating the conditions for the power generation. Figure 1 shows the NR profiles of Nafion at 80 °C at different relative humidities on SiO2 and Pt, respectively. Clear NR modulation was obtained under each condition on each specimen. The NR profiles on SiO2 and Pt were very different, showing different film morphologies. The characteristic bump around 0.2 Å-1 observed for Nafion on SiO2 (Fig. 1(a)) could be explained by the existence of a layer with a very different scattering length density (SLD) from that of Nafion, which was not observed for Nafion on Pt (Fig. 1(b)). The analyses of the NR profiles were carried out using the MOTOFIT program. For Nafion on SiO2, the NR data in Fig. 1(a) were fit very well with calculated values applying 4-layer models. Figure 2 shows the layered model of Nafion on SiO2/Si(100). SLD values were converted into Nafion and water densities at each sub-layer. The total thickness of the Nafion film at 80 ºC was 131, 139, 146, and 153 nm at 30, 50, 65, and 80% RH, respectively. The water uptake calculated from the NR data at 80% RH of a 100-nm Nafion on SiO2 was in good agreement with that measured by the mass of a film at 80 ºC.2) In “Topmost Layer” in Fig. 2, the water density was very low, and a hydrophobic conformation at the interface between Nafion and humidified N2 was proposed. The thickness of “Bulk Layer” monotonically increased from 120 to 140 nm as the humidity increased from 30 to 80% RH. The film thickness of “Intermediate Layer” slightly increased from 2.3 to 3.0 nm. The water density in “Intermediate Layer” was two times higher than that in “Bulk Layer”. The thickness of “Water-rich Layer” was 1.3 nm regardless of the humidity, while the water density greatly increased up to 0.97 g cm-3 at 80% RH. As seen in Fig. 1(b), no bump was observed on Nafion on Pt. The Nafion film on Pt/Si(100) was modeled with three layers with no “Water-rich Layer”. The influences of different substrates are clearly observed, which will be discussed in detail. This work was performed under the SPer-FC project of NEDO, Japan. The NR experiments were performed as projects approved by the Japan Proton Accelerator Research Complex under user programs, Nos. 2016A0246, 2016B0036, and 2017B0316. References 1) T. Kawamoto, M. Aoki, T. Kimura, T. Mizusawa, N. L. Yamada, J. Miyake, K. Miyatake, and J. Inukai, accepted to Jpn. J. Appl. Phys. 2) C. Yang, S. Srinivasan, A. B. Bocarsly, S. Tulyani, and J.B. Benziger, J. Membr. Sci. 237, 145, (2004). Figure 1

Etching gas-sieving nanopores in single-layer graphene with an angstrom precision for high-performance gas mixture separation.

One of the bottlenecks in realizing the potential of atom-thick graphene membrane for gas sieving is the difficulty in incorporating nanopores in an otherwise impermeable graphene lattice, with an angstrom precision at a high-enough pore density. We realize this design by developing a synergistic, partially decoupled defect nucleation and pore expansion strategy using O2 plasma and O3 treatment. A high density (ca. 2.1 × 1012 cm-2) of H2-sieving pores was achieved while limiting the percentage of CH4-permeating pores to 13 to 22 parts per million. As a result, a record-high gas mixture separation performance was achieved (H2 permeance, 1340 to 6045 gas permeation units; H2/CH4 separation factor, 15.6 to 25.1; H2/C3H8 separation factor, 38.0 to 57.8). This highly scalable pore etching strategy will accelerate the development of single-layer graphene-based energy-efficient membranes.

Transverse Kerker Scattering for Angstrom Localization of Nanoparticles.

Angstrom precision localization of a single nanoantenna is a crucial step towards advanced nanometrology, medicine, and biophysics. Here, we show that single nanoantenna displacements down to few angstroms can be resolved with sub-angstrom precision using an all-optical method. We utilize the tranverse Kerker scattering scheme where a carefully structured light beam excites a combination of multipolar modes inside a dielectric nanoantenna, which then, upon interference, scatters directionally into the far field. We spectrally tune our scheme such that it is most sensitive to the change in directional scattering per nanoantenna displacement. Finally, we experimentally show that antenna displacement down to 3Å is resolvable with a localization precision of 0.6Å.

Three-Dimensional Localization of an Individual Fluorescent Molecule with Angstrom Precision

Among imaging techniques, fluorescence microscopy is a unique method to noninvasively image individual molecules in whole cells. If the three-dimensional spatial precision is improved to the angstrom level, various molecular arrangements in the cell can be visualized on an individual basis. We have developed a cryogenic reflecting microscope with a numerical aperture of 0.99 and an imaging stability of 0.05 nm in standard deviation at a temperature of 1.8 K. The key optics to realize the cryogenic performances is the reflecting objective developed by our laboratory. With this cryogenic microscope, an individual fluorescent molecule (ATTO647N) at 1.8 K was localized with standard errors of 0.53 nm (x), 0.31 nm (y), and 0.90 nm (z) when 106 fluorescence photons from the molecule were accumulated in 5 min.

Multidomain structure and correlated dynamics determined by self-consistent FRET networks.

We present an approach that allows us to simultaneously access structure and dynamics of a multi-domain protein in solution. Dynamic domain arrangements are experimentally determined by combining self-consistent networks of distance distributions with known domain structures. Local structural dynamics are correlated with the global arrangements by analyzing networks of time-resolved single-molecule fluorescence parameters. The strength of this hybrid approach is shown by an application to the flexible multi-domain Hsp90. The average solution structure of Hsp90’s closed state resembles the known x-ray crystal structure with Angstrom precision. The open state is represented by an ensemble of conformations with inter-domain fluctuations of up to 25 Å. The data reveal a state-specific suppression of the sub-millisecond fluctuations by dynamic protein-protein interaction. Finally, the method enables localization and functional characterization of dynamic elements and domain interfaces.

Permanent matching of coupled optical bottle resonators with better than 0.16 GHz precision.

The fabrication precision is one of the most critical challenges to the creation of practical photonic circuits composed of coupled high Q-factor microresonators. While very accurate transient tuning of microresonators based on local heating has been reported, the record precision of permanent resonance positioning achieved by post-processing is still within 1 and 5 GHz. Here we demonstrate two coupled bottle microresonators fabricated at the fiber surface with resonances that are matched with a better than 0.16 GHz precision. This corresponds to a better than 0.17 Å precision in the effective fiber radius variation. The achieved fabrication precision is only limited by the resolution of our optical spectrum analyzer and can be potentially improved by an order of magnitude.

Single-Molecule FRET for Dynamic Structural Biology: DNA Polymerase i Structure and Mechanism with Angstrom Precision

A key question in the function of DNA-processing enzymes is their mechanism for rapid location and binding of target sites in DNA. DNA polymerase I (Pol) is involved in high-fidelity DNA replication and repair, and must recognize gapped and flapped DNA substrates. Due to their transient and dynamic nature, the structures of these complexes and the recognition mechanism by which they are formed are largely unknown.Combining single-molecule Forster Resonance Energy Transfer (smFRET) with dye-position modelling, we can obtain absolute distance measurements with angstrom precision. We have used 120 such distances in a rigid-body docking approach to determine the structure of the single-nucleotide-gapped DNA-Pol binary complex, and that of the gapped-DNA substrate alone.In the complex structure, the DNA substrate exhibits a 120-degree bend. The upstream DNA is channeled into the Pol active site, and its position is in excellent agreement (RMSD < 3 A) with the position of short DNA fragments seen in crystal structures of Pol. The downstream DNA is partially melted and held in close proximity to F771, a residue important for strand displacement.The structure of the gapped-DNA substrate alone adopts a 20-degree bend and a twist. Our coarse-grained simulations show short excursions of the substrate to partially kinked conformations not accessible to duplex DNA. Together, these data are consistent with a two-step model whereby Pol recognizes and binds a partially kinked conformation of gapped DNA, which then further rearranges to the fully bent state seen in the complex structure.Finally, using our method of internalization by electroporation, we have observed single-molecule FRET signals corresponding to unbound and Pol-bound gapped DNA in live bacteria, confirming the physiological relevance of the proposed substrate and complex structures and the recognition mechanism.

Nuclear magnetic resonance spectroscopy with single spin sensitivity.

Nuclear magnetic resonance spectroscopy and magnetic resonance imaging at the ultimate sensitivity limit of single molecules or single nuclear spins requires fundamentally new detection strategies. The strong coupling regime, when interaction between sensor and sample spins dominates all other interactions, is one such strategy. In this regime, classically forbidden detection of completely unpolarized nuclei is allowed, going beyond statistical fluctuations in magnetization. Here we realize strong coupling between an atomic (nitrogen–vacancy) sensor and sample nuclei to perform nuclear magnetic resonance on four 29Si spins. We exploit the field gradient created by the diamond atomic sensor, in concert with compressed sensing, to realize imaging protocols, enabling individual nuclei to be located with Angstrom precision. The achieved signal-to-noise ratio under ambient conditions allows single nuclear spin sensitivity to be achieved within seconds.

Cryogenic Colocalization Microscopy for Nanometer‐Distance Measurements

The main limiting factor in spatial resolution of localization microscopy is the number of detected photons. Recently we showed that cryogenic measurements improve the photostability of fluorophores, giving access to Angstrom precision in localization of single molecules. Here, we extend this method to colocalize two fluorophores attached to well-defined positions of a double-stranded DNA. By measuring the separations of the fluorophore pairs prepared at different design positions, we verify the feasibility of cryogenic distance measurement with sub-nanometer accuracy. We discuss the important challenges of our method as well as its potential for further improvement and various applications.

Molecular Dynamics of DNA–Protein Conjugates on Electrified Surfaces: Solutions to the Drift-Diffusion Equation

Self-assembled monolayers of charged polymers are an integral component of many state-of-the-art nanobiosensors. Electrical interactions between charged surfaces and charged biomolecules, adopting the roles of linkers or capture molecules, are not only crucial to the sensor performance but may also be exploited for novel sensing concepts based on electrically actuated interfaces. Here we introduce an analytical model describing the behavior of double-stranded DNA and proteins tethered to externally biased microelectrodes. Continuum electrostatic Poisson-Boltzmann models and the drift-diffusion (Smoluchowski) equation are used to calculate the steady state as well as the dynamic behavior of oligonucleotide rods in DC and AC electric fields. The model predicts the oligonucleotide orientation on the surface and calculates how the increased hydrodynamic drag caused by a protein bound to the DNA's distal end affects the molecular dynamics of the DNA-protein complex. The results of the model are compared to experiments with electrically switchable DNA layers, and very good agreement between theory and experiment is found. The hydrodynamic diameter of the bound protein can be analyzed from experimental data of the slowed motion of the DNA-protein conjugate with angstrom precision.