Sub-nanometer Resolution Research Articles

Diurnal variation of sun-induced chlorophyll fluorescence of agricultural crops observed from a point-based spectrometer on a UAV

Unmanned Aerial Vehicle (UAV)-based measurements allow studying sun-induced chlorophyll fluorescence (SIF) at the field scale and can potentially upscale results from ground to airborne/satellite level. The objective of this paper is to present the FluorSpec system providing SIF measurements at the field level onboard a UAV, and to evaluate the potential of this system for understanding diurnal SIF patterns for different arable crops. The core components of FluorSpec are a point spectrometer configured to measure in the O2 absorption bands at sub-nanometer resolution, bifurcated fibre optics to switch between the downwelling irradiance and upwelling radiance measurements, and a laser range finder allowing accurate atmospheric correction. The processing chain is explained and the capability of the novel Spectral Shape Assumption Fraunhofer Line Discrimination (SSA-FLD) method to retrieve SIF was tested. To test the reliability of FluorSpec diurnal SIF measurements, near-canopy diurnal SIF was monitored during the growing season over potato and sugar beet plants with a ground-based setup. The two crops exhibited a clear diurnal SIF pattern, which positively correlated with the photosynthetically active radiation (PAR). The divergence in diurnal patterns between SIF and PAR indicated that the crops might be suffering from heat stress. A significant correlation between SIF and the Photosystem II Quantum Yield was obtained. By mounting the FluorSpec on a UAV, SIF measurements were obtained over the same crops during a clear day. UAV-based SIF also exhibited a pronounced diurnal pattern similar to the ground-based measurements and it showed clear spatial variation within different crop fields. The obtained results demonstrate the ability of the FluorSpec system to reliably measure plant fluorescence at ground and field level, and the possibility of the UAV-based FluorSpec to bridge the scale gap between different levels of SIF observations.

Kilohertz frame rate snapshot hyperspectral imaging of metal reactive materials.

We demonstrate a kilohertz frame rate snapshot hyperspectral imaging system suitable for high-speed imaging, which we name snapshot hyperspectral imager for emission and reactions (SHEAR). This system splits the sensor of a single high-speed camera to simultaneously capture a conventional image and a spectrally sheared response of the scene under study. Given the small, point-source-like nature of burning metal micro-particles, the spectral response of the species is captured without the need for a slit, as is needed in conventional imaging spectrometers. We pair robust image registration techniques with sparse reconstruction algorithms to computationally disentangle overlapping spectra associated with many burning particles over the course of a combustion experiment. As a proof-of-concept experiment, representative physical vapor deposited Al:Zr composite particles are ignited, and their burn evolution is recorded at a frame rate of 2 kHz using this method. We demonstrate operation over two distinct wavelength ranges spanning hundreds of nanometers in wavelength and with sub-nanometer resolution. We are able to track hundreds of individual Al:Zr particles in a single high-speed video, providing ample statistics of burn time, temperature, and AlO emission timing in a high-throughput method. The demonstrated technology is high-throughput, flexible in wavelength, inexpensive, and relatively easy to implement, and provides a much needed tool for in situ composite metal fuel diagnostics.

Quantitative sampling of atomic-scale electromagnetic waveforms

Tailored nanostructures can confine electromagnetic waveforms in extremely sub-wavelength volumes, opening new avenues in lightwave sensing and control down to sub-molecular resolution. Atomic light–matter interaction depends critically on the absolute strength and the precise time evolution of the near field, which may be strongly influenced by quantum-mechanical effects. However, measuring atom-scale field transients has remained out of reach. Here we introduce quantitative atomic-scale waveform sampling in lightwave scanning tunnelling microscopy to resolve a tip-confined near-field transient. Our parameter-free calibration employs a single-molecule switch as an atomic-scale voltage standard. Although salient features of the far-to-near-field transfer follow classical electrodynamics, we develop a comprehensive understanding of the atomic-scale waveforms with time-dependent density functional theory. The simulations validate our calibration and confirm that single-electron tunnelling ensures minimal back-action of the measurement process on the electromagnetic fields. Our observations access an uncharted domain of nano-opto-electronics where local quantum dynamics determine femtosecond atomic near fields. Ultrafast lightwave sampling based on scanning tunnelling microscopy is developed to resolve near fields with sub-picosecond time resolution and sub-nanometre spatial resolution. Parameter-free quantitative measurement is achieved by using a single-molecule switch.

Atomic force microscopy for nanoscale mechanical property characterization

Over the past several decades, atomic force microscopy (AFM) has advanced from a technique used primarily for surface topography imaging to one capable of characterizing a range of chemical, mechanical, electrical, and magnetic material properties with subnanometer resolution. In this review, we focus on AFM as a nanoscale mechanical property characterization tool and examine various AFM contact and intermittent contact modes that add mechanical contrast to an imaged surface. Through detailed analysis of the tip-sample contact mechanics, this contrast can be converted into quantitative measurements of various nanomechanical properties including elastic modulus, shear modulus, wear rate, adhesion, and viscoelasticity. Different AFM modes that provide such measurements are compared and contrasted in this work on a wide range of materials including ceramics, metals, semiconductors, polymers, and biomaterials. In the last few years, considerable improvements have been made in terms of fast imaging capabilities, tip preservation, and quantitative mechanics for multifrequency measurements as well as well-known AFM modes like amplitude modulation and peak-force tapping. In line with these developments, a major highlight of this review is the discussion of the operation and capabilities of one such mode, namely, intermittent contact resonance AFM (ICR-AFM). The applications of ICR-AFM to nanoscale surface and subsurface quantitative mechanical characterizations are reviewed with specific examples provided for thin polymeric films and patterned nanostructures of organosilicate dielectric materials. The combination of AFM-based mechanical characterization with AFM-based chemical spectroscopy to allow nanoscale structure-property characterization is also discussed and demonstrated for the analysis of low-k dielectric/copper nanoelectronic interconnect structures and further highlights synergistic advances in the AFM field.

Ultra-short photoacoustic pulse generation through hot electron pressure in two-dimensional electron gas.

Launching ultrashort femtosecond photoacoustic pulses with multi-terahertz bandwidth will find broad applications from fundamental acoustics in 2D materials and THz-acoustic and phonon spectroscopy to nondestructive detection in opaque materials with a sub-nanometer resolution. Here we report the generation of ultra-short 344 fs photoacoustic pulses with a 2.1 THz bandwidth from interfacial two-dimensional electron gas using optical femtosecond excitation. A comparison with simulation supports the dominant contribution of hot electron pressure and the ultrafast electron relaxation to produce pulsewidth shorter than the acoustic transit time across the electron wavefunction. Our simulation further indicates the possibility to generate <200 fs photoacoustic pulse.

Sub-molecular photoluminescence

Photoluminescence spectroscopy using atomic-scale light reveals an optical transition of a single molecule at sub-nanometre resolution.

Holography: application to high-resolution imaging.

Electron holography was invented for correcting aberrations of the lenses of electron microscopes. It was used to observe the atomic arrangements in crystals after decades of research. Then it was combined with a hardware aberration corrector to enable high-resolution and high-precision analysis. Its applications were further extended to magnetic observations with sub-nanometer resolution. High-resolution electron holography has become a powerful technique for observing electromagnetic distributions in functional materials.

Sub-Nanometer Cryo-EM Density Map of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2.

Heterodimeric amino acid transporters (HATs) are protein complexes mediating the transport of amino acids and derivatives thereof across biological membranes. HATs are composed of two subunits, a heavy and a light chain subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. The human HAT 4F2hc-LAT2 is composed of the type-II membrane N-glycoprotein 4F2hc (SCL3A2) and the L-type amino acid transporter LAT2 (SLC7A8), which are covalently linked to each other by a conserved disulfide bridge. Whereas LAT2 catalyzes substrate transport, 4F2hc is important for the successful trafficking of the transporter to the plasma membrane. The overexpression, malfunction, or absence of 4F2hc-LAT2 is associated with human diseases, and therefore, this heterodimeric complex represents a potential drug target. The recombinant human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast Pichia pastoris, and the protein can be purified. Here, we present the cryo-EM density map of the human 4F2hc-LAT2 amino acid transporter at sub-nanometer resolution. A homology model of 4F2hc-LAT2 in the inward-open conformation was generated and fitted into the cryo-EM density and analyzed. In addition, disease-causing point mutations in human LAT2 were mapped on the homology model of 4F2hc-LAT2, and the possible functional implications on the molecular level are discussed.

Cryo-electron microscopy visualization of a large insertion in the 5S ribosomal RNA of the extremely halophilic archaeon Halococcus morrhuae.

The extreme halophile Halococcus morrhuae (ATCC® 17082) contains a 108-nucleotide insertion in its 5S rRNA. Large rRNA expansions in Archaea are rare. This one almost doubles the length of the 5S rRNA. In order to understand how such an insertion is accommodated in the ribosome, we obtained a cryo-electron microscopy reconstruction of the native large subunit at subnanometer resolution. The insertion site forms a four-way junction that fully preserves the canonical 5S rRNA structure. Moving away from the junction site, the inserted region is conformationally flexible and does not pack tightly against the large subunit. The high-salt requirement of the H. morrhuae ribosomes for their stability conflicted with the low-salt threshold for cryo-electron microscopy procedures. Despite this obstacle, this is the first cryo-electron microscopy map of Halococcus ribosomes.

Cryo-FIB-lift-out: practically impossible to practical reality.

In this paper, we explore the development of the Cryo-Lift-Out (cryo-LO) technique as preparation tool for cryogenic transmission electron microscopy (cryo-TEM). What started in early work defying what was considered 'practically impossible' has developed into state-of-the-art practical reality. This paper presents the key hardware, basic principles and key considerations for the practical usage of cryogenic Lift-Out for those new to the field. Detailed protocols and in-depth description of considerations and points for further development are presented. The authors have attempted to formalise everything known about the technique gathered together from their expertise gained in the development of this approach. LAY DESCRIPTION: A major challenge in electron microscopy is the production of suitable samples from hydrated biological and soft-matter materials for subnanometre resolution imaging in a cryo-Transmission Electron Microscope (TEM). A well-known solution for room temperature materials is called (in situ) Lift-Out. It uses a fine needle that picks up a tiny section called a lamella. Lamellae are made by a Focused Ion Beam (FIB). In this paper, we seek to set out the beginnings of Lift-Out sample preparation conducted under cryogenic conditions and the development of this approach as applied to frozen, hydrated biological and soft-matter samples. We discuss the required basic hardware and provide a thorough description of developed protocols. We aim at those new to the field of cryo-Lift-Out to fully educate them in the finer points of hardware setup and practical considerations when attempting to perform cryo-Lift-Out and to demonstrate what has been achieved thus far. We also discuss areas of further improvement and talking points for the future direction of this promising sample preparation technique.

An atomic force microscope integrated with a helium ion microscope for correlative nanoscale characterization

In this work, we report on the integration of an atomic force microscope (AFM) into a helium ion microscope (HIM). The HIM is a powerful instrument, capable of imaging and machining of nanoscale structures with sub-nanometer resolution, while the AFM is a well-established versatile tool for multiparametric nanoscale characterization. Combining the two techniques opens the way for unprecedented in situ correlative analysis at the nanoscale. Nanomachining and analysis can be performed without contamination of the sample and environmental changes between processing steps. The practicality of the resulting tool lies in the complementarity of the two techniques. The AFM offers not only true 3D topography maps, something the HIM can only provide in an indirect way, but also allows for nanomechanical property mapping, as well as for electrical and magnetic characterization of the sample after focused ion beam materials modification with the HIM. The experimental setup is described and evaluated through a series of correlative experiments, demonstrating the feasibility of the integration.

A review: additive manufacturing of flexure mechanism for nanopositioning system

A comprehensive review relevant to the design and fabrication of nanopositioning stages based on additive manufacturing (AM) technology has been for the first time introduced in the academic society. With the development of AM technology, AM has been applied in many engineering design areas such as aerospace, automotive, consumer electronics, and so on. Due to current limitations of AM tolerance (surface quality, form error), process obscurity (melting pool, layer adhesion), and cost (especially for metals or composites), there are only a small number of AM-applied devices that are currently available either in the market or in many industry sectors above. The flexure mechanisms that are typically employed in nanopositioning applications can provide a sub-nanometer resolution motion; however, their current manufacturing methods (milling, electric discharge machining, water jet machining) not only limit complicated flexure mechanisms in 3D geometries but also prevent designers from challenging novel topology optimizations. AM can overcome those limitations of fabrication and material distribution. Furthermore, it can allow new design approaches to topology optimizations. This review presents current and future AM-based precision motion device applications. Here, both design and fabrication of flexure mechanisms applied with current AM technology and the potential of further developments were discussed.

Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM

Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.

Design and Characterization of a Sharp GaAs/Zn(Mn)Se Heterovalent Interface: A Sub-Nanometer Scale View.

The distribution of magnetic impurities (Mn) across a GaAs/Zn(Mn)Se heterovalent interface is investigated combining three experimental techniques: Cross-Section Scanning Tunnel Microscopy (X-STM), Atom Probe Tomography (APT), and Secondary Ions Mass Spectroscopy (SIMS). This unique combination allowed us to probe the Mn distribution with excellent sensitivity and sub-nanometer resolution. Our results show that the diffusion of Mn impurities in GaAs is strongly suppressed; conversely, Mn atoms are subject to a substantial redistribution in the ZnSe layer, which is affected by the growth conditions and the presence of an annealing step. These results show that it is possible to fabricate a sharp interface between a magnetic semiconductor (Zn(Mn)Se) and high quality GaAs, with low dopant concentration and good optical properties.

Refining internal bilayer structure of bicelles resolved by extended-q small angle X-ray scattering

The internal profile across the bilayer reveals important structural information regarding the crystallinity of acyl chains or the positions of encapsulated species. Here, we demonstrate that a simple five-layer-core-shell discoidal model can be employed to best fit the extended-q small angle X-ray scattering (SAXS) data and resolve the bilayer internal structure (with sub-nanometer resolution) of a nanoscale discoidal system comprised of a mixture of long- and short- chain lipids (known as “bicelles”). In contrast to the traditional core-shell discoidal model, the detailed structure in the hydrophobic core such as the methylene and methyl groups can be distinguished via this model. The refined model is validated by the SAXS data of bicelles whose electron scattering length density of the hydrophobic core is adjusted by the addition of a long-chain lipid with a fluorine-end group. The higher resolution of the bilayer internal structure can be employed to advance our understanding of the interaction and conformation of the membrane and associated molecules, such as membrane-associated proteins and locations of entrapped species in the lipid nanoparticles.

A fiber scanning tunneling microscope for optical analysis at the nanoscale.

A hybrid scanning tunneling/optical near-field microscope is presented, in which an optical fiber tip coated with 100 nm thick Ag/Cr films scans the surface. The tip metallization enables operating the instrument via a current-based distance control and guarantees sub-nanometer spatial resolution in the topographic channel. The fiber tip simultaneously serves as nanoscale light source, given the optical transparency of the metal coating. The emission response of the tip-sample junction is collected with two parabolic mirrors and probed with a far-field detector. To test the capabilities of the new setup, the evolution of the optical signal is monitored when the tip approaches a gold surface. The intensity rise and frequency shift of the emission provide evidence for the development of coupled plasmon modes in the tip-sample cavity. Photon mapping is employed to probe the optical inhomogeneity of Ru(0001) and TiO2(110) surfaces covered with silver deposits. While the 2D Ag flakes on Ru give rise to a near-field enhancement, the 3D particles on titania locally damp the gap plasmons and lower the emitted intensity. The lateral resolution in the optical channel has been estimated to be ∼1 nm, and optical and topographic signals are well correlated. Our fiber microscope thus appears to be suitable for probing optical surface properties at the nanoscale.

Impact of substrate on tip-enhanced Raman spectroscopy: A comparison between field-distribution simulations and graphene measurements

Tip-enhanced Raman spectroscopy (TERS) has reached nanometer spatial resolution for measurements performed at ambient conditions and sub-nanometer resolution at ultra high vacuum. Super-resolution (beyond the tip apex diameter) TERS has been obtained, mostly in the gap mode configuration, where a conductive substrate localizes the electric fields. Here we present experimental and theoretical TERS to explore the field distribution responsible for spectral enhancement. We use gold tips of $40\pm 10 \ \text{nm}$ apex diameter to measure TERS on graphene, a spatially delocalized two-dimensional sample, sitting on different substrates: (i) glass, (ii) a thin layer of gold and (iii) a surface covered with $12\ \text{nm}$ diameter gold spheres, for which $6\ \text{nm}$ resolution is achieved at ambient conditions. The super-resolution is due to the field configuration resulting from the coupled tip-sample-substrate system, exhibiting a non-trivial spatial surface distribution. The field distribution and the symmetry selection rules are different for non-gap vs. gap mode configurations. This influences the overall enhancement which depends on the Raman mode symmetry and substrate structure.

Subnanometer structures of HIV-1 envelope trimers on aldrithiol-2-inactivated virus particles.

The HIV-1 envelope glycoprotein (Env) trimer, composed of gp120 and gp41 subunits, mediates viral entry into cells. Recombinant Env trimers have been studied structurally, but characterization of Env embedded in intact virus membranes has been limited to low resolution. Here, we deploy cryo-electron tomography and subtomogram averaging to determine the structures of Env trimers on aldrithiol-2 (AT-2)-inactivated virions in ligand-free, antibody-bound and CD4-bound forms at subnanometer resolution. Tomographic reconstructions document molecular features consistent with high-resolution structures of engineered soluble and detergent-solubilized Env trimers. One of three conformational states previously predicted by smFRET was not observed by cryo-ET, potentially owing to AT-2 inactivation. We did observe Env trimers to open in situ in response to CD4 binding, with an outward movement of gp120-variable loops and an extension of a critical gp41 helix. Overall features of Env trimer embedded in AT-2-treated virions appear well-represented by current engineered trimers.

Probing Magnetism in Artificial Metal-Organic Complexes Using Electronic Spin Relaxometry.

Single spins are considered as a versatile candidate for miniaturizing information devices down to the nanoscale. To engineer the spin's properties, metal-organic frameworks provide a promising route which in turn requires thorough understanding of the metal-molecule interaction. Here, we investigate the magnetic robustness of a single iron (Fe) atom in artificially built Fe-tetracyanoethylene (TCNE) complexes by using low-temperature scanning tunneling microscopy (STM). We find that the magnetic anisotropy and spin relaxation dynamics of the Fe atom within the complexes remain unperturbed in comparison to well-isolated Fe atoms. Density functional theory (DFT) calculations support our experimental findings, suggesting that the 3d orbitals of the Fe atom remain largely undisturbed while the 4s and 4p orbitals are rearranged in the process of forming a complex. To precisely determine the location of the spin center within the complex, we utilize STM-based spin relaxometry, mapping out the spatial dependence of spin relaxation with subnanometer resolution. Our work suggests that the magnetic properties of atoms can remain unchanged while being embedded in a weakly bound molecular framework.

Towards high-throughput in situ structural biology using electron cryotomography

Electron cryotomography is a rapidly evolving method for imaging macromolecules directly within the native environment of cells and tissues. Combined with sub-tomogram averaging, it allows structural and cell biologists to obtain sub-nanometre resolution structures in situ. However, low throughput in cryo-ET sample preparation and data acquisition, as well as difficulties in target localisation and sub-tomogram averaging image processing, limit its widespread usability. In this review, we discuss new advances in the field that address these throughput and technical problems. We focus on recent efforts made to resolve issues in sample thinning, improvement in data collection speed at the microscope, strategies for localisation of macromolecules using correlated light and electron microscopy and advancements made to improve resolution in sub-tomogram averaging. These advances will considerably decrease the amount of time and effort required for cryo-ET and sub-tomogram averaging, ushering in a new era of structural biology where in situ macromolecular structure determination will be routine.