High-resolution Topographic Images Research Articles

Machine learning approaches for improving atomic force microscopy instrumentation and data analytics

Atomic force microscopy (AFM) is a part of the scanning probe microscopy family. It provides a platform for high-resolution topographical imaging, surface analysis as well as nanomechanical property mapping for stiff and soft samples (live cells, proteins, and other biomolecules). AFM is also crucial for measuring single-molecule interaction forces and important parameters of binding dynamics for receptor-ligand interactions or protein-protein interactions on live cells. However, performing AFM measurements and the associated data analytics are tedious, laborious experimental procedures requiring specific skill sets and continuous user supervision. Significant progress has been made recently in artificial intelligence (AI) and deep learning (DL), extending into microscopy. In this review, we summarize how researchers have implemented machine learning approaches so far to improve the performance of atomic force microscopy (AFM), make AFM data analytics faster, and make data measurement procedures high-throughput. We also shed some light on the different application areas of AFM that have significantly benefited from applications of machine learning frameworks and discuss the scope and future possibilities of these crucial approaches.

Design and performance of an ultrahigh vacuum spectroscopic-imaging scanning tunneling microscope with a hybrid vibration isolation system.

A spectroscopic imaging-scanning tunneling microscope (SI-STM) allows for the atomic scale visualization of the surface electronic and magnetic structure of novel quantum materials with a high energy resolution. To achieve the optimal performance, a low vibration facility is required. Here, we describe the design and performance of an ultrahigh vacuum STM system supported by a hybrid vibration isolation system that consists of a pneumatic passive and a piezoelectric active vibration isolation stage. We present the detailed vibrational noise analysis of the hybrid vibration isolation system, which shows that the vibration level can be suppressed below 10-8 m/sec/√Hz for most frequencies up to 100Hz. Combined with a rigid STM design, vibrational noise can be successfully removed from the tunneling current. We demonstrate the performance of our STM system by taking high resolution spectroscopic maps and topographic images on several quantum materials. Our results establish a new strategy to achieve an effective vibration isolation system for high-resolution STM and other scanning probe microscopies to investigate the nanoscale quantum phenomena.

Aeromagnetic surveys for the location of undocumented orphaned wells

The U.S. Department of Energy's National Energy Technology Laboratory (NETL) conducted research to field test the ability of airborne methods to locate existing wells quickly and accurately with minimal landowner impact. Helicopter surveys with two boom-mounted atomic magnetometers were used to locate wells at two oil fields in Wyoming and at four large oil and gas producing areas in Pennsylvania. At the two Wyoming oil fields, the helicopter magnetic survey identified 91%–96% of the wells located by exhaustive ground search. A different validation was used for the thickly forested Pennsylvania flight areas where well picks from the helicopter magnetic survey were used to direct the ground search to potential well locations for verification. The number and location of verified well locations were then compared to well records in the state database. For the oldest wells (circa 1860–1880), the number of wells in the state database exceeded the number of wells identified by the helicopter magnetic survey because many early wells had wood casing (nonmagnetic). For wells drilled between 1880 and 1950, the helicopter surveys found two to five undocumented wells for each well in the state database. The best results were achieved at a gas storage field where wells were drilled after 1950. There, 98% of the wells located by the helicopter magnetic survey were documented. In 2017, NETL used an atomic magnetometer on a small uncrewed aircraft system (sUAS) to refly an area previously flown with a crewed helicopter. The magnetic map from the sUAS survey was equal to that obtained with a crewed helicopter. High-resolution topographic images developed from sUAS LiDAR surveys were found to be helpful in locating very early wells with wood casing. When the wood casing decayed, soil subsided into the well void, leaving a circular depression that is easily recognized in high-resolution LiDAR imagery.

Unmanned Aerial Vehicle Derived DEM Using in Accurate Geometric Analysis for Water Harvesting in Small-Scale Depressions

Due to significant progress in remote sensing over the past few decades, topographic data is now widely available on a global scale. Recent advancements in unmanned aerial vehicle technology have made it possible to get high resolution topographic images on a scale of less than a meter. Making it perfect for small sites survey. Conducted an assessment of the unmanned aerial vehicle ability to estimate various geometric elements by generating a digital elevation model and subsequently analyzing the depression geometrically. The study area in focus was in AL-Sherqat, which consists of a small valley. Upon analysis of the generated digital elevation model, we were able to determine the valley's maximum capacity volume to be 18365.91 m3 at a water level of 186.8 m above sea level. The analysis also yielded other important elements, such as positive volume, negative and positive surface areas, negative and positive planar areas, residual capacity, average depression depth, and average island thickness. The interrelationships among these geometric elements were plotted for further analysis. Based on the results, we conclude that the unmanned aerial vehicle structure from the motion (SFM) algorithm and the produced digital elevation model are suitable for surveying small topographic depressions. This technique could prove valuable for water harvesting purposes.

Recent advances in the application of atomic force microscopy to structural biology

The application of atomic force microscopy (AFM) for functional imaging and manipulating biomolecules at all levels of organization has enabled great progress in the structural biology field over the last decades, contributing to the discovery of novel structural entities of biological significance across many disciplines ranging from biochemistry, biomedicine and biophysics to molecular and cell biology, up to food systems and beyond. AFM has the capability to generate high-resolution topographic images spanning from the submolecular to the (sub)cellular range and can probe biochemical and biophysical sample properties in close to native conditions with excellent temporal resolution. Instrumental developments in the past decade enable dynamical structural and conformational studies of single biomolecules and new techniques for structural and chemical modification of the AFM probe have converted the cantilever into a versatile tool to study different biological phenomena, such as the mechanical stability of biomolecular complexes or the force induced dynamic changes of mechanically stressed proteins at the nanoscopic level. To improve the functionality of AFM and approach dynamic processes of complex biological systems ex vivo, AFM is combined with complementary microscopy, nanoscopy and spectroscopy tools. These multimethodological approaches provide unprecedented possibilities of probing physical, chemical and biological properties of complex cellular systems with high spatio-temporal resolution, leading to novel applications that correlate structural results with functional biochemical, biophysical, immunological, or genetic data of the system under study.

Sharp, high numerical aperture (NA), nanoimprinted bare pyramid probe for optical mapping.

The ability to correlate optical hyperspectral mapping and high resolution topographic imaging is critically important to gain deep insight into the structure-function relationship of nanomaterial systems. Scanning near-field optical microscopy can achieve this goal, but at the cost of significant effort in probe fabrication and experimental expertise. To overcome these two limitations, we have developed a low-cost and high-throughput nanoimprinting technique to integrate a sharp pyramid structure on the end facet of a single-mode fiber that can be scanned with a simple tuning-fork technique. The nanoimprinted pyramid has two main features: (1) a large taper angle (∼70°), which determines the far-field confinement at the tip, resulting in a spatial resolution of 275nm, an effective numerical aperture of 1.06, and (2) a sharp apex with a radius of curvature of ∼20nm, which enables high resolution topographic imaging. Optical performance is demonstrated through evanescent field distribution mapping of a plasmonic nanogroove sample, followed by hyperspectral photoluminescence mapping of nanocrystals using a fiber-in-fiber-out light coupling mode. Through comparative photoluminescence mapping on 2D monolayers, we also show a threefold improvement in spatial resolution over chemically etched fibers. These results show that the bare nanoimprinted near-field probes provide simple access to spectromicroscopy correlated with high resolution topographic mapping and have the potential to advance reproducible fiber-tip-based scanning near-field microscopy.

Deep Learning for Live Cell Shape Detection and Automated AFM Navigation.

Atomic force microscopy (AFM) provides a platform for high-resolution topographical imaging and the mechanical characterization of a wide range of samples, including live cells, proteins, and other biomolecules. AFM is also instrumental for measuring interaction forces and binding kinetics for protein–protein or receptor–ligand interactions on live cells at a single-molecule level. However, performing force measurements and high-resolution imaging with AFM and data analytics are time-consuming and require special skill sets and continuous human supervision. Recently, researchers have explored the applications of artificial intelligence (AI) and deep learning (DL) in the bioimaging field. However, the applications of AI to AFM operations for live-cell characterization are little-known. In this work, we implemented a DL framework to perform automatic sample selection based on the cell shape for AFM probe navigation during AFM biomechanical mapping. We also established a closed-loop scanner trajectory control for measuring multiple cell samples at high speed for automated navigation. With this, we achieved a speed-up in AFM navigation and reduced the time involved in searching for the particular cell shape in a large sample. Our innovation directly applies to many bio-AFM applications with AI-guided intelligent automation through image data analysis together with smart navigation.

Revealing the distribution of active submarine faults off the coast of Oga Peninsula using high-resolution stereoscopic topographic images

This study aims to elucidate the importance of a combination of tectonic geomorphological interpretations using high-resolution stereoscopic topographic images and seismic profile interpretations as methodological standards for organizing maps of the distribution of active submarine faults. Furthermore, we provide topographic evidence of active submarine reverse faults off the coast of the Oga Peninsula, which lies on the eastern margin of the Japan Sea. First, the study reprocessed the point cloud data of multi-beam bathymetry data and produced a new digital bathymetry model and anaglyph images. These images and contour maps enabled the identification of broad deformation and other small-scale tectonic landforms, such as fault scarp, fault scarp on the flat surface of a basin, and flexure scarp. We first described detailed tectonic landforms along the NE–SW trending northwest-dipping faults across the Mogami Trough, which was identified by previous geological studies, to demonstrate the effectiveness of the proposed method. Moreover, we applied the method as an applicable example for tectonic landforms in the east-dipping fault at the offshore of the Oga Peninsula with seismic profile interpretation. The result demonstrated that an active fault across the central part of the Mogami Trough can be recognized on the bases of geology and topography. However, identifying an active fault that extends to the area with poor sediment deposition at the northern end of the trough is difficult. The study found that detecting active fault distribution by tectonic geomorphological interpretation is practical and effective using a stereoscopic image from high-resolution topographical data. Moreover, a new east-dipping reverse fault, that is, the Kyurokujima-nansei-oki fault, was discovered along the eastern margin of the northwestern part of the Mogami Trough by identifying flexure scarp and comparison with seismic profiles. The strike of the Kyurokujima-nansei-oki fault is parallel to that of the aftershock distribution of the southwest part of the 1983 Central Japan Sea Earthquake and resembles an east-dipping structure. Thus, this fault should be considered one of the source faults during assessments of seismic and tsunami hazards.

High-Resolution Topographic and Chemical Surface Imaging of Chalk for Oil Recovery Improvement Applications

Chalk is a very fine-grained carbonate and can accommodate high porosity which is a key characteristic for high-quality hydrocarbon reservoirs. A standard procedure within Improved Oil Recovery (IOR) is seawater-injection which repressurizes the reservoir pore pressure. Long-term seawater-injection will influence mineralogical processes as dissolution and precipitation of secondary minerals. These secondary minerals (<1 micrometer) precipitate during flooding experiments mimicking reservoir conditions. Due to their small sizes, analysis from traditional scanning electron microscopy combined with energy dispersive X-ray spectroscopy is not conclusive because of insufficient spatial resolution and detection limit. Therefore, chalk was analyzed with high-resolution imaging by helium ion microscopy (HIM) combined with secondary ion mass spectrometry (SIMS) for the first time. Our aim was to identify mineral phases at sub-micrometer scale and identify locations of brine–rock interactions. In addition, we wanted to test if current understanding of these alteration processes can be improved with the combination of complementary imaging techniques and give new insights to IOR. The HIM-SIMS imaging revealed well-defined crystal boundaries and provided images of excellent lateral resolution, allowing for identification of specific mineral phases. Using this new methodology, we developed chemical identification of clay minerals and could define their exact location on micron-sized coccolith grains. This shows that it is essential to study mineralogical processes at nanometer scale in general, specifically in the research field of applied petroleum geology within IOR.

Enhancement of graphene-related and substrate-related Raman modes through dielectric layer deposition

In this report, we demonstrate a method for the enhancement of Raman active modes of hydrogen-intercalated quasi-free-standing epitaxial chemical vapor deposition graphene and the underlying semi-insulating 6H–SiC(0001) substrate through constructive signal interference within atomic-layer-deposited amorphous Al2O3 passivation. We find that an optimum Al2O3 thickness of 85 nm for the graphene 2D mode and one of 82 nm for the SiC longitudinal optical A1 mode at 964 cm–1 enable a 60% increase in their spectra intensities. We demonstrate the method's efficiency in Raman-based determination of the dielectric thickness and high-resolution topographic imaging of a graphene surface.

Study of Amyloid Fibers Using Atomic Force Microscopy.

Atomic force microscopy (AFM) provides high-resolution images of the topography of amyloid fibers adsorbed on surfaces. This information is very useful to study their molecular assembly under various conditions. This chapter describes the basic protocols required to deposit fibers on flat surfaces and discusses some of the practical issues required to operate a good commercial microscope setup to obtain appropriate high-resolution AFM topographic images of amyloid fibers.

Reconstituted virus-nucleus system reveals mechanics of herpesvirus genome uncoating.

The viral replication cycle is controlled by information transduced through both molecular and mechanical interactions. Viral infection mechanics remains largely unexplored, however, due to the complexity of cellular mechanical responses over the course of infection as well as a limited ability to isolate and probe these responses. Here, we develop an experimental system consisting of herpes simplex virus type 1 (HSV-1) capsids bound to isolated and reconstituted cell nuclei, which allows direct probing of capsid-nucleus mechanics with atomic force microscopy (AFM). Major mechanical transformations occur in the host nucleus when pressurised viral DNA ejects from HSV-1 capsids docked at the nuclear pore complexes (NPCs) on the nuclear membrane. This leads to structural rearrangement of the host chromosome, affecting its compaction. This in turn regulates viral genome replication and transcription dynamics as well as the decision between a lytic or latent course of infection. AFM probing of our reconstituted capsid-nucleus system provides high-resolution topographical imaging of viral capsid docking at the NPCs as well as force volume mapping of the infected nucleus surface, reflecting mechanical transformations associated with chromatin compaction and stiffness of nuclear lamina (to which chromatin is tethered). This experimental system provides a novel platform for investigation of virus-host interaction mechanics during viral genome penetration into the nucleus.

High-resolution compositional mapping of surfaces in non-contact atomic force microscopy by a new multi-frequency excitation

In this paper, a new multi-frequency excitation method based on combination resonance is introduced to enhance the non-contact atomic force microscopy performance. In combination resonance, excitation frequencies are selected so that summation/subtraction of excitation frequencies is close to the natural frequencies of the microcantilever. Due to the nonlinear nature of this method, the probe response to excitation is very sensitive to change in tip-sample forces. This could be used to generate high-resolution compositional mapping and topographical images of the surface. The present study reveals that both amplitude and phase shift of the combination resonance are sensitive to change in parameters such as Hamaker constant, damping coefficient, Young’s modulus and tip-sample initial distance. It is observed that because of high sensitivity to Hamaker constant a small change in the surface material leads to considerable variations in amplitude and phase shift. This sensitivity is employed to improve compositional mapping of the surface materials. It is also found out that the response amplitude in the combination resonance is very sensitive to change in the tip-sample initial distance. This sensitivity may be used to reduce the vertical noise and increase image resolution, especially in environments with low quality factors. Overall, using this technique the image contrast increases significantly and high resolution compositional mapping of surfaces is achieved.

AFM-Based Correlative Microscopy Illuminates Human Pathogens.

Microbes have an arsenal of virulence factors that contribute to their pathogenicity. A number of challenges remain to fully understand disease transmission, fitness landscape, antimicrobial resistance and host heterogeneity. A variety of tools have been used to address diverse aspects of pathogenicity, from molecular host-pathogen interactions to the mechanisms of disease acquisition and transmission. Current gaps in our knowledge include a more direct understanding of host-pathogen interactions, including signaling at interfaces, and direct phenotypic confirmation of pathogenicity. Correlative microscopy has been gaining traction to address the many challenges currently faced in biomedicine, in particular the combination of optical and atomic force microscopy (AFM). AFM, generates high-resolution surface topographical images, and quantifies mechanical properties at the pN scale under physiologically relevant conditions. When combined with optical microscopy, AFM probes pathogen surfaces and their physical and molecular interaction with host cells, while the various modes of optical microscopy view internal cellular responses of the pathogen and host. Here we review the most recent advances in our understanding of pathogens, recent applications of AFM to the field, how correlative AFM-optical microspectroscopy and microscopy have been used to illuminate pathogenicity and how these methods can reach their full potential for studying host-pathogen interactions.

A Non-Destructive, Tuneable Method to Isolate Live Cells for High-Speed AFM Analysis.

Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever tip, and not negatively affect the sample surface for data acquisition. Here, we describe an inherently flexible methodology, utilising the high-resolution three-dimensional based printing technique of multiphoton polymerisation to rapidly generate bespoke arrays for cellular AFM analysis. As an example, we present data collected from live Emiliania huxleyi cells, unicellular microalgae, imaged by contact mode High-Speed Atomic Force Microscopy (HS-AFM), including one cell that was imaged continuously for over 90 min.

A horizontal-type scanning near-field optical microscope with torsional mode operation toward high-resolution and non-destructive imaging of soft materials.

We design and build a horizontal-type aperture based scanning near-field optical microscope (a-SNOM) with superior mechanical stability toward high-resolution and non-destructive topographic and optical imaging. We adopt the torsional mode in AFM (atomic force microscopy) operation to achieve a better force sensitivity and a higher topographic resolution when using pyramidal a-SNOM tips. The performance and stability of the AFM are evaluated through single-walled carbon nanotube and poly(3-hexyl-thiophene) nanowire samples. An optical resolution of 93 nm is deduced from the a-SNOM imaging of a metallic grating. Finally, a-SNOM fluorescence imaging of soft lipid domains is successfully achieved without sample damage by our horizontal-type a-SNOM instrument with torsional mode AFM operation.

Experimental study on molecular fluctuations of biomolecules by force spectroscopy

Atomic force microscopy (AFM) measurements of biomolecules, such as proteins, are often affected by molecular fluctuations. Not only due to the intrinsic thermal fluctuation, deformation and displacement of a part or the whole molecule are induced by the AFM tip, especially when the molecules are weakly fixed to a substrate. Here we have studied the effect of the molecular fluctuations on the topography of the streptavidin (SA) protein molecules bound to a DNA origami in two different configurations by force spectroscopy experiments. While the experimental and theoretical height histograms of a tightly-fixed SA molecule were in good agreement, the experimental height histogram of a weakly-fixed SA molecule was distorted. We discussed the origins of the discrepancy of the histogram for the latter and suggested that it was due to the tip-induced deformation or the displacement of the molecule. The methodology presented here is useful for assessment of the appropriate fixation conditions for high-resolution topographic and functional imaging experiments of a single molecule by AFM.

Mechanical properties measured by atomic force microscopy define health biomarkers in ageing C. elegans

Genetic and environmental factors are key drivers regulating organismal lifespan but how these impact healthspan is less well understood. Techniques capturing biomechanical properties of tissues on a nano-scale level are providing new insights into disease mechanisms. Here, we apply Atomic Force Microscopy (AFM) to quantitatively measure the change in biomechanical properties associated with ageing Caenorhabditis elegans in addition to capturing high-resolution topographical images of cuticle senescence. We show that distinct dietary restriction regimes and genetic pathways that increase lifespan lead to radically different healthspan outcomes. Hence, our data support the view that prolonged lifespan does not always coincide with extended healthspan. Importantly, we identify the insulin signalling pathway in C. elegans and interventions altering bacterial physiology as increasing both lifespan and healthspan. Overall, AFM provides a highly sensitive technique to measure organismal biomechanical fitness and delivers an approach to screen for health-improving conditions, an essential step towards healthy ageing.

Imaging low-dimensional nanostructures by very low voltage scanning electron microscopy: ultra-shallow topography and depth-tunable material contrast

We demonstrate the implications of very low voltage operation (<1 kV) of a scanning electron microscope for imaging low-dimensional nanostructures where standard voltages (2–5 kV) involve a beam penetration depth comparable to the cross-section of the nanostructures. In this common situation, image sharpness, contrast quality and resolution are severely limited by emission of secondary electrons far from the primary beam incidence point. Oppositely, very low voltage operation allows reducing the beam-specimen interaction to an extremely narrow and shallow region around the incidence point, enabling high-resolution and ultra-shallow topographic contrast imaging by high-angle backscattered electrons detection on the one hand, and depth-tunable material contrast imaging by low-angle backscattered electrons detection on the other. We describe the performance of these imaging approaches on silicon nanowires obtained by the vapor-liquid-solid mechanism. Our experimental results, supported by Monte Carlo simulations of backscattered electrons emission from the nanowires, reveal the self-assembly of gold-silica core-shell nanostructures at the nanowire tips without any ad-hoc thermal oxidation step. This result demonstrates the capacity of very low voltage operation to provide optimum sharpness, contrast and resolution in low-dimensional nanostructures and to gather information about nanoscaled core-shell conformations otherwise impossible to obtain by standard scanning electron microscopy alone.

Assessment of biophysical properties of Haemonchus contortus from different life cycle stages with atomic force microscopy

Atomic force microscopy (AFM) was used in this work to investigate the ultrastructural and mechanical characteristics of Haemonchus contortus, the major gastrointestinal nematode that infects small ruminants worldwide. The biophysical characterization of this species is extremely important in order to reveal mechanisms of action of drugs and to classify its ultrastructure and biomechanical properties. High-resolution topographic images by AFM as well as data on biomechanical properties of cuticles were obtained at different stages of H. contortus. The results reveal details of the mechanical and structural properties of this nematode never observed before for nematodes parasite with other microscope techniques. Qualitative and quantitative reductions in the elasticity of the larvae stage egg were compared with those of the morulae stage, and the increased adhesion of unsheathed L3 were compared with the same stage of sheathed larvae. The results presented here open possibilities for understanding the mechanisms of drug and biomolecular actions that can be used to control infections caused by H. contortus.