Nanometer Spatial Resolution Research Articles

Introduction to Optical Tweezers: Background, System Designs, and Applications.

Optical tweezers are a means to manipulate objects with light. With the technique, microscopically small objects can be held and steered, allowing for accurate measurement of the forces applied to these objects. Optical tweezers can typically obtain a nanometer spatial resolution, a picoNewton force resolution, and a millisecond time resolution, which makes the technique well suited for the study of biological processes from the single-cell down to the single-molecule level. In this chapter, we aim to provide an introduction to the use of optical tweezers for single-molecule analyses. We start from the basic principles and methodology involved in optical trapping, force calibration, and force measurements. Next, we describe the components of an optical tweezers setup and their experimental relevance. Finally, we will provide an overview of the broad applications in context of biological research, with the emphasis on the measurement modes, experimental assays, and possible combinations with fluorescence microscopy techniques.

Mid-infrared photocurrent nano-spectroscopy exploiting the thermoelectric effect in graphene

We develop a conductive-atomic force microscope coupled to a mid-infrared (IR) quantum cascade laser that allows to measure mid-IR photocurrent maps and spectra with nanometric spatial resolution. Here, we exploit the photo-induced thermoelectric effect of graphene to obtain the mid-IR local absorption spectra of nanoscale devices and materials. By scanning the probe in AFM-contact mode, the thermoelectric photocurrent can be mapped throughout the graphene layer, which acts as a semitransparent transducer. At zero voltage bias applied to the probe tip, we observe very low noise (about 25 pA/Hz), which makes our technique competitive with others in measuring the local IR absorption in nanomaterials with insufficient thermal expansion coefficient, or to characterize nanoscale devices that significantly absorb IR radiation, such as mid-infrared photodetectors.

Magneto-optical diffraction of visible light as a probe of nanoscale displacement of domain walls at femtosecond timescales.

Using diffraction of femtosecond laser pulses of visible light by a magnetic domain pattern in an iron garnet, we demonstrate a proof of concept of time-resolved measurements of domain pattern movements with nanometer spatial and femtosecond temporal resolution. In this method, a femtosecond laser (pump) pulse initiates magnetization dynamics in a sample that is initially in a labyrinth domain state, while an equally short linearly polarized laser pulse (probe) is diffracted by the domain pattern. The components of the diffracted light that are polarized orthogonally to the incident light generate several concentric diffraction rings. Nanometer small changes in the relative sizes of domains with opposite magnetization result in observable changes in the intensities of the rings. We demonstrate that the signal-to-noise ratio is high enough to detect a 6nm domain wall displacement with 100fs temporal resolution using visible light. We also discuss possible artifacts, such as pump-induced changes of optical properties, that can affect the measurements.

Ultrastructural features mirror metabolic derangement in human endothelial cells exposed to high glucose

High glucose-induced endothelial dysfunction is the early event that initiates diabetes-induced vascular disease. Here we employed Cryo Soft X-ray Tomography to obtain three-dimensional maps of high d-glucose-treated endothelial cells and their controls at nanometric spatial resolution. We then correlated ultrastructural differences with metabolic rewiring. While the total mitochondrial mass does not change, high d-glucose promotes mitochondrial fragmentation, as confirmed by the modulation of fission–fusion markers, and dysfunction, as demonstrated by the drop of membrane potential, the decreased oxygen consumption and the increased production of reactive oxygen species. The 3D ultrastructural analysis also indicates the accumulation of lipid droplets in cells cultured in high d-glucose. Indeed, because of the decrease of fatty acid β-oxidation induced by high d-glucose concentration, triglycerides are esterified into fatty acids and then stored into lipid droplets. We propose that the increase of lipid droplets represents an adaptive mechanism to cope with the overload of glucose and associated oxidative stress and metabolic dysregulation.

Neural network-assisted single-molecule localization microscopy with a weak-affinity protein tag

Single-molecule localization microscopy achieves nanometer spatial resolution by localizing single fluorophores separated in space and time. A major challenge of single-molecule localization microscopy is the long acquisition time, leading to low throughput, as well as to a poor temporal resolution that limits its use to visualize the dynamics of cellular structures in live cells. Another challenge is photobleaching, which reduces information density over time and limits throughput and the available observation time in live-cell applications. To address both challenges, we combine two concepts: first, we integrate the neural network DeepSTORM to predict super-resolution images from high-density imaging data, which increases acquisition speed. Second, we employ a direct protein label, HaloTag7, in combination with exchangeable ligands (xHTLs), for fluorescence labeling. This labeling method bypasses photobleaching by providing a constant signal over time and is compatible with live-cell imaging. The combination of both a neural network and a weak-affinity protein label reduced the acquisition time up to ∼25-fold. Furthermore, we demonstrate live-cell imaging with increased temporal resolution, and capture the dynamics of the endoplasmic reticulum over extended time without signal loss.

Dynamic mapping of proteome trafficking within and between living cells by TransitID

The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.

Effect of Ni substitution on the antiferromagnetic domains of cobalt oxide

We present a spatially resolved X-ray magnetic linear dichroism study of high quality micron-sized mixed nickel-cobalt oxide (NCO) crystals. NixCo1−xO was prepared in-situ by high-temperature oxygen-assisted molecular beam epitaxy on a Ru(0001) single crystal substrate. To check the effect of incorporating Ni into the cobalt oxide films, three different compositions were prepared. The element-specific XMLD measurements reveal strong antiferromagnetic contrast at room temperature and magnetic domains up to one micron in size, reflecting the high structural quality of the NCO islands. By means of vectorial magnetometry, the antiferromagnetic spin axis orientation of the domains was determined with nanometer spatial resolution, and found to depend on the stoichiometry of the prepared crystals.

Plasmonic Nanopyramid Array Enhancing Luminescence of MoS2 Investigated by Cathodoluminescence

AbstractTransition metal dichalcogenides (TMDs) like molybdenum disulfide (MoS2) have unique optical properties such as strong luminescence and a tunable bandgap. However, the small interaction distance due to the atomically thin nature of these materials is a limitation for applications. In this study, a significant enhancement of the A‐ and B‐exciton emission in MoS2 flakes through coupling with plasmonic Au nano‐pyramid arrays fabricated by a colloidal lithography technique is reported. This study experimentally maps local emission enhancement from the structure with a nanometer spatial resolution by cathodoluminescence (CL) technique. Strongly enhanced emission intensity within the MoS2‐Au pyramids is achieved due to the localized surface plasmon resonance confined in nano‐pyramids and its interaction with the periodicity‐based resonance of the array structure. The results provide an approach to significantly improve the luminescence intensity of MoS2 through optical interaction with the local plasmonic field, which is promising for applications in optoelectronic devices and nanophotonics.

Antibody binding reports spatial heterogeneities in cell membrane organization

The spatial organization of cell membrane glycoproteins and glycolipids is critical for mediating the binding of ligands, receptors, and macromolecules on the plasma membrane. However, we currently do not have the methods to quantify the spatial heterogeneities of macromolecular crowding on live cell surfaces. In this work, we combine experiment and simulation to report crowding heterogeneities on reconstituted membranes and live cell membranes with nanometer spatial resolution. By quantifying the effective binding affinity of IgG monoclonal antibodies to engineered antigen sensors, we discover sharp gradients in crowding within a few nanometers of the crowded membrane surface. Our measurements on human cancer cells support the hypothesis that raft-like membrane domains exclude bulky membrane proteins and glycoproteins. Our facile and high-throughput method to quantify spatial crowding heterogeneities on live cell membranes may facilitate monoclonal antibody design and provide a mechanistic understanding of plasma membrane biophysical organization.

Insights into the Dynamics and Helicase Activity of RecD2 of Deinococcus radiodurans during DNA Repair: A Single-Molecule Perspective.

While the double helix is the most stable conformation of DNA inside cells, its transient unwinding and subsequent partial separation of the two complementary strands yields an intermediate single-stranded DNA (ssDNA). The ssDNA is involved in all major DNA transactions such as replication, transcription, recombination, and repair. The process of DNA unwinding and translocation is shouldered by helicases that transduce the chemical energy derived from nucleotide triphosphate (NTP) hydrolysis to mechanical energy and utilize it to destabilize hydrogen bonds between complementary base pairs. Consequently, a comprehensive understanding of the molecular mechanisms of these enzymes is essential. In the last few decades, a combination of single-molecule techniques (force-based manipulation and visualization) have been employed to study helicase action. These approaches have allowed researchers to study the single helicase-DNA complex in real-time and the free energy landscape of their interaction together with the detection of conformational intermediates and molecular heterogeneity during the course of helicase action. Furthermore, the unique ability of these techniques to resolve helicase motion at nanometer and millisecond spatial and temporal resolutions, respectively, provided surprising insights into their mechanism of action. This perspective outlines the contribution of single-molecule methods in deciphering molecular details of helicase activities. It also exemplifies how each technique was or can be used to study the helicase action of RecD2 in recombination DNA repair.

High-Yield Deterministic Focused Ion Beam Implantation of Quantum Defects Enabled by In Situ Photoluminescence Feedback.

Focused ion beam implantation is ideally suited for placing defect centers in wide bandgap semiconductors with nanometer spatial resolution. However, the fact that only a few percent of implanted defects can be activated to become efficient single photon emitters prevents this powerful capability to reach its full potential in photonic/electronic integration of quantum defects. Here an industry adaptive scalable technique is demonstrated to deterministically create single defects in commercial grade silicon carbide by performing repeated low ion number implantation and in situ photoluminescence evaluation after each round of implantation. An array of 9 single defects in 13 targeted locations is successfully created-a ≈70% yield which is more than an order of magnitude higher than achieved in a typical single pass ion implantation. The remaining emitters exhibit non-classical photon emission statistics corresponding to the existence of at most two emitters. This approach can be further integrated with other advanced techniques such as in situ annealing and cryogenic operations to extend to other material platforms for various quantum information technologies.

Evaluation protocol for revealing magnonic contrast in TR-STXM measurements

We present a statistically motivated method to extract magnonic contrast from time-resolved scanning transmission x-ray microscopy (TR-STXM) measurements. TR-STXM is an element-specific method for resolving spin-dynamics in space and time. It offers nanometer spatial resolution and picosecond temporal resolution. The presented method makes it possible to obtain phase and amplitude profiles of spin-waves from STXM measurements. Furthermore, it allows for a rigorous transformation to reciprocal magnon k⃗-space, revealing k⃗-dependent magnon properties such as the magnon dispersion in three dimensions and for all directions of the magnetic anisotropy. We demonstrate our method using X-band ferromagnetic resonance on a micrometer-sized permalloy assembly.

Plasmonic near-field spatiotemporal characterizations of an asymmetric copper bowtie nanostructure

Photon-induced near-field electron microscopy (PINEM), developed from ultrafast transmission electron microscopy, enables near-field imaging with nanometer spatial resolution and femtosecond temporal resolution. We report the plasmonic near-field distribution and lifetime analysis for an asymmetric copper bowtie nanostructure having unequal opening angles. The PINEM images show surface plasmon polaritons and local surface-plasmon resonance excitations with various polarizations. Combined with finite-element simulations, the polarization- and structure-dependent distribution and intensity variations of the near-field were analyzed. The lifetime difference of a plasmonic near-field excited by different polarizations is also discussed. The temporal and spatial characterization of the plasmonic near-field is important for the further studies of plasmonic near-field manipulation and designs of plasmonic devices having specific functions.

Direct observation of motor protein stepping in living cells using MINFLUX.

Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but these measurements have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in two and three dimensions using the recently introduced super-resolution technique MINFLUX. Using this approach, we resolved the precise stepping motion of the motor protein kinesin-1 as it walked on microtubules in living cells. Nanoscopic tracking of motors walking on the microtubules of fixed cells also enabled us to resolve the architecture of the microtubule cytoskeleton with protofilament resolution.

Extending Imaging Volume in Soft X‐Ray Tomography

Soft X‐ray tomography offers rapid imaging of whole, single cells with a few tens of nanometers spatial resolution without fixation or labeling. Herein, this technique is limited to specimens about 10 μm thick, such that applications of soft X‐ray tomography of large human cells or multicellular specimens are not possible. A theoretical and experimental framework for soft X‐ray tomography that enables extension of imaging volumes to 18 μm‐thick specimens is developed. This approach, based on long depth of field and half‐acquisition tomography, is easily applicable to microscopes equipped with a full‐rotation specimen stage. This opens opportunities for imaging large human cells, such as those commonly seen in cancer research, as well as cell‐to‐cell interactions, where two or more cells occupy the same imaging volume.

Plasmon-Enhanced Raman Scattering by Multilayered Graphene at the Micro- and Nanoscale: SERS and TERS Analysis

This work is devoted to the study of plasmon-enhanced Raman scattering by the fundamental vibrational modes of multilayered graphene films. The film thickness was ∼3.5 nm, which corresponds to ∼10 monolayers. Multilayered graphene films were placed on a plasmonic substrate consisting of arrays of gold nanodisks (50–250 nm in diameter). Surface-enhanced Raman scattering by the main vibrational modes of multilayered graphene film placed on an array of Au nanodisks of various sizes was implemented. The measurements were performed at excitation wavelengths of 532, 638, and 785 nm. A resonant SERS enhancement of the main vibrational modes of multilayered graphene by a factor of 25 was achieved for nanodisks with a diameter of 103 nm upon excitation at 638 nm. A stronger local enhancement of Raman scattering in multilayered graphene (by a factor of 50) placed on Au nanodisk array is achieved using gap-mode tip-enhanced Raman scattering (gap-mode TERS). Nanofolds in the graphene film appeared due to the corrugated surface of the plasmonic substrate were visualized with nanometer spatial resolution. It is shown that the frequency positions of G and 2D modes of nanofolds decrease with respect to the corresponding values in flat multilayered graphene manifesting mechanical stresses in the nanofolds up to 0.7%. The results obtained shed light on the effects of the interaction of multilayered graphene with metal nanostructures and are important in creating hybrid metal/graphene plasmonic substrates.

MAXPEEM: a spectromicroscopy beamline at MAX IV laboratory.

MAXPEEM, a dedicated photoemission electron microscopy beamline at MAX IV Laboratory, houses a state-of-the-art aberration-corrected spectroscopic photoemission and low-energy electron microscope (AC-SPELEEM). This powerful instrument offers a wide range of complementary techniques providing structural, chemical and magnetic sensitivities with a single-digit nanometre spatial resolution. The beamline can deliver a high photon flux of≥1015 photons s-1 (0.1% bandwidth)-1 in the range 30-1200 eV with full control of the polarization from an elliptically polarized undulator. The microscope has several features which make it unique from similar instruments. The X-rays from the synchrotron pass through the first beam separator and impinge the surface at normal incidence. The microscope is equipped with an energy analyzer and an aberration corrector which improves both the resolution and the transmission compared with standard microscopes. A new fiber-coupled CMOS camera features an improved modulation transfer function, dynamic range and signal-to-noise ratio compared with the traditional MCP-CCD detection system.

Nanoscale mapping of point defect concentrations with 4D-STEM

Vacancies are missing atoms in a crystalline material, and occur both at equilibrium (varying with temperature) and out of equilibrium such as when crystalline materials are damaged with radiation or corrosion. While we know of their importance, particularly regarding diffusive mechanisms, it is not straightforward to experimentally measure their concentration or to visualize them directly. Traditionally, measurements such as positron annihilation spectroscopy and to some degree X-ray diffraction can measure average concentrations, but generally lack the ability to visualize or quantify a heterogenous concentration of vacancies that can occur at the level of individual defects and microstructural features. Here, we present a method to map vacancy concentrations and their distribution using local lattice parameter measurements with a high-resolution electron microscope. Our method utilizes a Au thin film as a model to demonstrate the method via four-dimensional scanning transmission electron microscopy (4D-STEM) by correlating the differences between changes in lattice parameter and the volumetric thermal expansion during in situ heating experiments. The vacancy mapping methodology is also applied to non-equilibrium defects accumulated in pure Al via knock-on electron beam irradiation. Our method demonstrates the ability to map point defect concentrations in heterogeneous systems in situ with nanometer spatial resolution. The result is a technique that can provide direct measurements of vacancy concentrations at the level of individual defects in studies of materials in and out of equilibrium.

Electron Microscopy Studies of Soft Nanomaterials.

This review highlights recent efforts on applying electron microscopy (EM) to soft (including biological) nanomaterials. We will show how developments of both the hardware and software of EM have enabled new insights into the formation, assembly, and functioning (e.g., energy conversion and storage, phonon/photon modulation) of these materials by providing shape, size, phase, structural, and chemical information at the nanometer or higher spatial resolution. Specifically, we first discuss standard real-space two-dimensional imaging and analytical techniques which are offered conveniently by microscopes without special holders or advanced beam technology. The discussion is then extended to recent advancements, including visualizing three-dimensional morphology of soft nanomaterials using electron tomography and its variations, identifying local structure and strain by electron diffraction, and recording motions and transformation by in situ EM. On these advancements, we cover state-of-the-art technologies designed for overcoming the technical barriers for EM to characterize soft materials as well as representative application examples. The even more recent integration of machine learning and its impacts on EM are also discussed in detail. With our perspectives of future opportunities offered at the end, we expect this review to inspire and stimulate more efforts in developing and utilizing EM-based characterization methods for soft nanomaterials at the atomic to nanometer length scales in academic research and industrial applications.

Quantum sensing of strongly coupled light-matter systems using free electrons.

Strong coupling in light-matter systems is a central concept in cavity quantum electrodynamics and is essential for many quantum technologies. Especially in the optical range, full control of highly connected multi-qubit systems necessitates quantum coherent probes with nanometric spatial resolution, which are currently inaccessible. Here, we propose the use of free electrons as high-resolution quantum sensors for strongly coupled light-matter systems. Shaping the free-electron wave packet enables the measurement of the quantum state of the entire hybrid systems. We specifically show how quantum interference of the free-electron wave packet gives rise to a quantum-enhanced sensing protocol for the position and dipole orientation of a subnanometer emitter inside a cavity. Our results showcase the great versatility and applicability of quantum interactions between free electrons and strongly coupled cavities, relying on the unique properties of free electrons as strongly interacting flying qubits with miniscule dimensions.