High-resolution Atomic Force Microscopy Research Articles

High-Resolution Atomic Force Microscopy Imaging of RNA Molecules in Solution.

Atomic force microscopy (AFM) is an important and versatile technique to investigate the structures and dynamics of biomolecules under physiologically relevant conditions at the single-molecule level. Recent progresses in high-resolution AFM imaging of nucleic acids have expanded this technique from simple characterization of double-stranded DNA or RNA to detailed analyses of the structure and dynamics of large functional RNAs with complex folds. Several technical developments, such as sharper probes and more stable instruments with novel imaging modes, AFM is capable of directly visualizing RNA conformational heterogeneity in solution in real time. Here, we introduce a comprehensive method for recording high-resolution images of RNA molecules, including sample preparation, instrument setup, data acquisition, and image processing.

High-resolution atomic force microscopy as a tool for topographical mapping of surface budding.

Extracellular vesicles (EVs) are membranous nanoparticles secreted by almost all cell types. Reflecting the physiopathological state of the parental cell, EVs circulate in all body fluids, reaching distant cell targets and delivering different bioactive cargoes. As biological carriers, EVs influence their microenvironment altering cellular responses, being considered promising biomarkers for both physiological and pathological conditions. EVs are heterogeneous in terms of size and composition, depending on cell type and exposure to stimuli, and different methods have been developed to characterize their morphological, biophysical, and biochemical features. Among them, electron microscopy (EM) is the main technique used, however, the lack of standardized protocols makes it difficult to characterize EVs with a good reproducibility, thus using multiple approaches may represent a way to obtain more precise information. Furthermore, the relationship between architecture and function, not only in a molecular, but also in a cellular level, is gaining growing emphasis, characterizing morphometric parameters may represent a distinct, but effective approach to study the physiopathological state of the cell. Atomic force microscopy (AFM), may represent a promising method to study in detail EVs dynamics throughout the cell surface and its variations related to the physiological state, overcoming the limits of EM, and providing more reliable information. In this study, human neuroblastoma SH-SY5Y cell line, a cellular model to investigate neurodegeneration and oxidative stress, has been used to perform a comparative morphological and quantitative analysis of membrane budding and isolated large vesicles-enriched (microvesicles-like vesicles; MVs) fraction from control or oxidative stressed cells. Our main goal was to build up a methodology to characterize EVs morphology and spatial distribution over the cell surface in different physiological conditions, and to evaluate the efficacy of AFM against conventional EM. Interestingly, both microscopy techniques were effective for this analysis, but AFM allowed to reveal a differential profiling of plasma membrane budding between the physiological and the stress condition, indicating a potential relationship between mechanical characteristics and functional role. The results obtained may provide interesting perspectives for the use of AFM to study EVs, validating a morphometric approach to understand the pathophysiological state of the cell related to EVs trafficking.

Micro-nano morphology parameters and mechanical properties of soot particles sampled from high pressure jet flames of diesel from direct coal liquefaction

Diesel from direct coal liquefaction (DDCL) is a new type of engine alternative energy. In this study, a premixed constant volume combustion chamber system with soot particle sampling devices were built. High resolution transmission electron microscope (HR-TEM) and atomic force microscopy were applicated to characterize the micro-nano morphology parameters and mechanical properties of soot particles sampled from high pressure jet flames of DDCL. There were two in-house automatic processing codes were developed to process the HR-TEM images and extract the micro-nano morphology parameters of the soot particles. This study has systematically studied the effect of injection pressures on the micro-nano morphology parameters and mechanical properties of soot particles. The test results illustrated that there was a correlation between the micro-nano morphology parameters and mechanical properties of soot particles. Under the same combustion conditions, DDCL soot particles demonstrated smaller size, compacted structure, lower graphitization degree and weaker antioxidant capacity than that of diesel soot particles. The average adhesive force and energy dissipation of diesel soot particles were much higher, which indicated the diesel soot particles have stronger agglomeration performance. The average elastic modulus of DDCL soot particles was smaller than that of diesel, which caused the weaker ability to resist deformation for DDCL soot particles.

Aging study on BiFe0.95Mn0.05O3 ferroelectric thin films with different preferred orientations

The BiFe0.95Mn0.05O3 (BFMO) thin films were epitaxial grown on the SrRuO3/LaAlO3 (SRO/LAO) single crystal substrates with different orientations (<100 >,<110 >and<111 >) by sol-gel and layer-by-layer rapid thermal annealing methods, respectively. The test techniques including X-ray diffraction (XRD), Raman, high resolution field emission scanning electron microscope (HR-SEM) and atomic force microscope (AFM) were applied to analyze the structure of the film samples. The results showed that a two-phase coexistence structure grown in all three films with the rhombohedral R3c as the main phase and the orthogonal Pnma as the second phase. The PFM tests revealed that the <111> oriented sample had normal island domain configuration, while the <100> and <110> oriented samples mainly grew non-180° domains. By comparing the aging behavior of the three samples after a room temperature period of aging for 30 and 120 days, it was believed that the film samples with different orientations appeared distinct aging degrees due to the different number of polarized equivalent components along <111> direction and the different angle between the equivalent components and the applied electric field direction. In particular, the film sample with <111> orientation showed almost no aging. It was also believed that the defect dipole pinning happened in the ferroelectric domain bodies would play a leading role in the aging process.

3D Generation of Multipurpose Atomic Force Microscopy Tips (Adv. Sci. 27/2022)

3D Generation of Multipurpose Atomic Force Microscopy Tips In article number 2201489, Ayoub Glia, Muhammedin Deliorman, and Mohammad A. Qasaimeh report on the technology of 3D generation of multipurpose atomic force microscopy tips (3DTIPs). 3DTIPs are developed with great flexibility in material, design, integration, and function. 3DTIPs are capable of obtaining high resolution and high-speed atomic force microscope imaging using common modes, well suited for various applications in air and liquid, and can significantly stimulate advances in various fields. 3DTIPs might serve as a milestone in advancing scanning probe microscopy from a mire common imaging tool towards an evolved platform integrable with broader research applications.

Bacterial surface interactions with organic colloidal particles: Nanoscale hotspots of organic matter in the ocean.

Colloidal particles constitute a substantial fraction of organic matter in the global ocean and an abundant component of the organic matter interacting with bacterial surfaces. Using E. coli ribosomes as model colloidal particles, we applied high-resolution atomic force microscopy to probe bacterial surface interactions with organic colloids to investigate particle attachment and relevant surface features. We observed the formation of ribosome films associating with marine bacteria isolates and natural seawater assemblages, and that bacteria readily utilized the added ribosomes as growth substrate. In exposure experiments ribosomes directly attached onto bacterial surfaces as 40-200 nm clusters and patches of individual particles. We found that certain bacterial cells expressed surface corrugations that range from 50-100 nm in size, and 20 nm deep. Furthermore, our AFM studies revealed surface pits in select bacteria that range between 50-300 nm in width, and 10-50 nm in depth. Our findings suggest novel adaptive strategies of pelagic marine bacteria for colloid capture and utilization as nutrients, as well as storage as nanoscale hotspots of DOM.

Native architecture and acclimation of photosynthetic membranes in a fast-growing cyanobacterium.

Efficient solar energy conversion is ensured by the organization, physical association, and physiological coordination of various protein complexes in photosynthetic membranes. Here, we visualize the native architecture and interactions of photosynthetic complexes within the thylakoid membranes from a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) using high-resolution atomic force microscopy. In the Syn2973 thylakoid membranes, both photosystem I (PSI)-enriched domains and crystalline photosystem II (PSII) dimer arrays were observed, providing favorable membrane environments for photosynthetic electron transport. The high light (HL)-adapted thylakoid membranes accommodated a large amount of PSI complexes, without the incorporation of iron-stress-induced protein A (IsiA) assemblies and formation of IsiA-PSI supercomplexes. In the iron deficiency (Fe-)-treated thylakoid membranes, in contrast, IsiA proteins densely associated with PSI, forming the IsiA-PSI supercomplexes with varying assembly structures. Moreover, type-I NADH dehydrogenase-like complexes (NDH-1) were upregulated under the HL and Fe- conditions and established close association with PSI complexes to facilitate cyclic electron transport. Our study provides insight into the structural heterogeneity and plasticity of the photosynthetic apparatus in the context of their native membranes in Syn2973 under environmental stress. Advanced understanding of the photosynthetic membrane organization and adaptation will provide a framework for uncovering the molecular mechanisms of efficient light harvesting and energy conversion.

High-Resolution Atomic Force Microscopy Study of Protein Surface Conformations

High-Resolution Atomic Force Microscopy Study of Protein Surface Conformations

3D Generation of Multipurpose Atomic Force Microscopy Tips

In this work, 3D polymeric atomic force microscopy (AFM) tips, referred to as 3DTIPs, are manufactured with great flexibility in design and function using two‐photon polymerization. With the technology holding a great potential in developing next‐generation AFM tips, 3DTIPs prove effective in obtaining high‐resolution and high‐speed AFM images in air and liquid environments, using common AFM modes. In particular, it is shown that the 3DTIPs provide high‐resolution imaging due to their extremely low Hamaker constant, high speed scanning rates due to their low quality factor, and high durability due to their soft nature and minimal isotropic tip wear; the three important features for advancing AFM studies. It is also shown that refining the tip end of the 3DTIPs by focused ion beam etching and by carbon nanotube inclusion substantially extends their functionality in high‐resolution AFM imaging, reaching angstrom scales. Altogether, the multifunctional capabilities of 3DTIPs can bring next‐generation AFM tips to routine and advanced AFM applications, and expand the fields of high speed AFM imaging and biological force measurements.

Molecule graph reconstruction from atomic force microscope images with machine learning

Despite the success of noncontact atomic force microscopy (AFM) in providing atomic-scale insight into the structure and properties of matter on surfaces, the wider applicability of the technique faces challenges in the difficulty of interpreting the measurement data. We tackle this problem by proposing a machine learning model for extracting molecule graphs of samples from AFM images. The predicted graphs contain not only atoms and their bond connections but also their coordinates within the image and elemental identification. The model is shown to be effective on simulated AFM images, but we also highlight some issues with robustness that need to be addressed before generalization to real AFM images.Impact statementDeveloping better techniques for imaging matter at the atomic scale is important for advancing our fundamental understanding of physics and chemistry as well as providing better tools for materials R&D of nanotechnologies. State-of-the-art high-resolution atomic force microscopy experiments are providing such atomic-resolution imaging for many systems of interest. However, greater automation of processing the measurement data is required in order to eliminate the need for subjective evaluation by human operators, which is unreliable and requires specialized expertise. The ability to convert microscope images into graphs would provide an easily understandable and precise view into the structure of the system under study. Furthermore, a graph consisting of a discrete set of objects, rather than an image that describes a continuous domain, is much more amenable to further processing and analysis using symbolic reasoning based on physically motivated rules. This type of image-to-graph conversion is also relevant to other machine learning tasks such as scene understanding.Graphical abstract

On-Surface Synthesis of Unsaturated Hydrocarbon Chains through C-S Activation.

We use an on‐surface synthesis approach to drive the homocoupling reaction of a simple dithiophenyl‐functionalized precursor on Cu(111). The C−S activation reaction is initiated at low annealing temperature and yields unsaturated hydrocarbon chains interconnected in a fully conjugated reticulated network. High‐resolution atomic force microscopy imaging reveals the opening of the thiophenyl rings and the presence of trans‐ and cis‐oligoacetylene chains as well as pentalene units. The chemical transformations were studied by C 1s and S 2p core level photoemission spectroscopy and supported by theoretical calculations. At higher annealing temperature, additional cyclization reactions take place, leading to the formation of small graphene flakes.

High resolution atomic force microscopy with an active piezoelectric microcantilever.

Active microcantilevers with on-chip sensing and actuation provide significant advantages in tapping mode Atomic Force Microscopy (AFM). Collocated transduction allows for effective manipulation of cantilever dynamics through feedback control, enabling higher scan rates. However, the adjacency of the sensing and actuation electrodes is known to result in a high level of feedthrough, leading to a low imaging resolution. Readout circuit noise further deteriorates the imaging precision. Here, we investigate the noise sources that affect AFM microcantilevers with collocated aluminum nitride (AlN) actuator-sensor pairs. We reported these cantilevers in earlier work and demonstrated that they display a very low level of feedthrough between the actuation and sensing electrodes. We present a high signal-to-noise ratio (SNR) sensing method that enables us to demonstrate high-resolution AFM on a calibration grating with nm-step silicon carbide (SiC) terraces. Measuring the Lorentzian response of the cantilever's Brownian motion with the on-chip active sensor at resonance enables us to calibrate the dynamic stiffness at the first fundamental resonance mode, without utilizing an optical sensor.

Submolecular Resolution Imaging of P3HT:PCBM Nanostructured Films by Atomic Force Microscopy: Implications for Organic Solar Cells.

The efficiency of organic bulk-heterojunction (BHJ) solar cells depends greatly on both the bulk and surface structure of the nanostructured bicontinuous interpenetrating network of materials, known as the active layer. The morphology of the top layer of a coated film is often resolved at the scale of a few nanometers, but fine details of the domains and the order within them are more difficult to identify. Here, we report a high-resolution atomic force microscopy (AFM) investigation of various stoichiometries of the well-studied poly(3-hexylthiophene):[6,6]-phenyl C61 butyric acid methyl ester (P3HT:PCBM) active layer mixture. Images of the surface were obtained using AC-mode AFM exciting higher-order resonance frequencies of a standard silicon probe, a promising technique for acquiring real-space images of organic-based thin films with nanoscale and even submolecular resolution. We provide firm evidence of the nanoscale organization of the P3HT polymer and of the P3HT:PCBM stoichiometric mixtures at the surface-air interface of the BHJ architecture. Our study shows the characteristic periodicity of the regioregular P3HT identified in the nanoscale domain areas with submolecular resolution. Such areas are then distorted in place when adding different quantities of PCBM forming stoichiometric mixtures. When the samples were exposed to ambient light, the morphologies were very different, and submolecular resolution was not achieved. This approach is shown to provide a precise view of the active layer's nanostructure and will be useful for studies of other materials as a function of various parameters, with particular attention to the role of the acceptor in tuning morphology for understanding optimum performance in organic photovoltaic devices.

Gas cluster etching for the universal preparation of polymer composites for nano chemical and mechanical analysis with AFM

Atomic force microscopy (AFM) based techniques that probe nanomechanical and nanochemical features are valuable tools for studying composite surfaces at high-resolution. However, the application of AFM, particularly at resolutions less than 50 nm per pixel, is limited to sample surfaces that are smooth relative to the tip dimensions and damage-free. As a result, high-resolution AFM is limited in technologically relevant polymer composite systems where current sample preparation techniques can fail to produce the necessary pristine surfaces. Here, we apply gas cluster ion beam (GCIB) etching to prepare composite materials for nanomechanical and nanochemical analysis. We demonstrate that the GCIB can effectively remove material and produce damage free surfaces for composites comprised of PMMA or PS confined in the 7 nm diameter pores of an organosilicate matrix. AFM-IR measurements on the etched region indicate a high degree of hydrogen bonding between the carbonyl groups of the confined PMMA and the hydroxyl groups on the pore walls, while maps of loss tangent can identify the 7 nm polymer domains within the organosilicate matrix. This work demonstrates that GCIB is an effective tool for nanocomposite surface preparation and could enable a new range of materials to be studied with AFM at high resolution.

Ultrastructural characterisation of young and aged dental enamel by atomic force microscopy.

Recent advances in atomic force microscopy (AFM) have allowed the characterisation of dental-associated biomaterials and biological surfaces with high resolution. In this context, the topography of dental enamel - the hardest mineralised tissue in the body - has been explored with AFM-based approaches at the microscale. With age, teeth are known to suffer changes that can impact their structural stability and function; however, changes in enamel structure because of ageing have not yet been explored with nanoscale resolution. Therefore, the aim of this exploratory work was to optimise an approach to characterise the ultrastructure of dental enamel and determine potential differences in topography, hydroxyapatite (HA) crystal size, and surface roughness at the nanoscale associated to ageing. For this, a total of six teeth were collected from human donors from which enamel specimens were prepared. By employing intermittent contact (AC mode) imaging, HA crystals were characterised in both transversal and longitudinal orientation (respect to surface plane) with high resolution in environmental conditions. The external enamel surface displayed the presence of a pellicle-like coating on its surface that was not observable on cleaned specimens. Acid-etching exposed crystals that were imaged and morphologically characterised in high resolution at the nanoscale in both the external and internal regions of enamel in older and younger specimens. Our results demonstrated important individual variations in HA crystal width and roughness parameters across the analysed specimens; however, an increase in surface roughness and decrease in HA width was observed for the pooled older external enamel group compared to younger specimens. Overall, high-resolution AFM was an effective approach for the qualitative and quantitative characterisation of human dental enamel ultrastructure. Future work should focus on exploring the ageing of dental enamel with increased sample sizes to compensate for individual differences as well as other potential confounding factors such as behavioural habits and mechanical forces.

Probing the Structural Details of Chitin Nanocrystal–Water Interfaces by Three‐Dimensional Atomic Force Microscopy

Chitin is one of the most abundant and renewable natural biopolymers. It exists in the form of crystalline microfibrils and is the basic structural building block of many biological materials. Its surface crystalline structure is yet to be reported at the molecular level. Herein, atomic force microscopy (AFM) in combination with molecular dynamics simulations reveals the molecular-scale structural details of the chitin nanocrystal (chitin NC)-water interface. High-resolution AFM images reveal the molecular details of chitin chain arrangements at the surfaces of individual chitin NCs, showing highly ordered, stable crystalline structures almost free of structural defects or disorder. 3D-AFM measurements with submolecular spatial resolution demonstrate that chitin NC surfaces interact strongly with interfacial water molecules creating stable, well-ordered hydration layers. Inhomogeneous encapsulation of the underlying chitin substrate by these hydration layers reflects the chitin NCs' multifaceted surface character with different chain arrangements and molecular packing. These findings provide important insights into chitin NC structures at the molecular level, which is critical for developing the properties of chitin-based nanomaterials. Furthermore, these results will contribute to a better understanding of the chemical and enzymatic hydrolysis of chitin and other native polysaccharides, which is also essential for the enzymatic conversion of biomass.

Growth of Fe-BDC Metal-Organic Frameworks onto Functionalized Si (111) Surfaces.

The realization of metal-organic framework (MOF) layers onto solid surfaces is a prerequisite for their integration into devices. This work reports the direct growth of Fe3+ /benzene di-carboxylate MOFs onto functionalized silicon surfaces, compatible with a wide range of MOF synthesis conditions. The co-nucleation and growth of different crystalline phases are evidenced, whose coverage depends on the surface chemistry and/or the solution composition. Three structural phases - the cubic MIL-101(Fe), a hexagonal phase with a structure close to MOF-235 and a MIL-53(Fe) with a monoclinic symmetry - are identified through characteristic crystal shapes and their structural parameters inferred from X-Ray Diffraction. In addition to the oriented growth of 3D crystallites, the formation of two-dimensional MIL-101 nano-crystallites or thin layers/islands exhibiting extended monocrystalline domains with (111) texture is also demonstrated through high-resolution atomic force microscopy. Post-synthesis treatments reveal a weak adhesion of the hexagonal phase, indicating a different surface anchoring.

Gasdermin-A3 pore formation propagates along variable pathways

Gasdermins are main effectors of pyroptosis, an inflammatory form of cell death. Released by proteolysis, the N-terminal gasdermin domain assembles large oligomers to punch lytic pores into the cell membrane. While the endpoint of this reaction, the fully formed pore, has been well characterized, the assembly and pore-forming mechanisms remain largely unknown. To resolve these mechanisms, we characterize mouse gasdermin-A3 by high-resolution time-lapse atomic force microscopy. We find that gasdermin-A3 oligomers assemble on the membrane surface where they remain attached and mobile. Once inserted into the membrane gasdermin-A3 grows variable oligomeric stoichiometries and shapes, each able to open transmembrane pores. Molecular dynamics simulations resolve how the membrane-inserted amphiphilic β-hairpins and the structurally adapting hydrophilic head domains stabilize variable oligomeric conformations and open the pore. The results show that without a vertical collapse gasdermin pore formation propagates along a set of multiple parallel but connected reaction pathways to ensure a robust cellular response.

The articular cartilage surface is impaired by a loss of thick collagen fibers and formation of type I collagen in early osteoarthritis

Osteoarthritis (OA) is a joint disease affecting millions of patients worldwide. During OA onset and progression, the articular cartilage is destroyed, but the underlying complex mechanisms remain unclear. Here, we uncover changes in the thickness of collagen fibers and their composition at the onset of OA. For articular cartilage explants from knee joints of OA patients, we find that type I collagen-rich fibrocartilage-like tissue was formed in macroscopically intact cartilage, distant from OA lesions. Importantly, the number of thick fibers (>100 nm) has decreased early in the disease, followed by complete absence of thick fibers in advanced OA. We have obtained these results by a combination of high-resolution atomic force microscopy imaging under near-native conditions, immunofluorescence, scanning electron microscopy and a fluorescence-based classification of the superficial chondrocyte spatial organization. Taken together, our data suggests that the loss of tissue functionality in early OA cartilage is caused by a reduction of thick type II collagen fibers, likely due to the formation of type I collagen-rich fibrocartilage, followed by the development of focal defects in later OA stages. We anticipate that such an integrative characterization will be very beneficial for an in-depth understanding of other native biological tissues and the development of sustainable biomaterials. Statement of significanceIn early osteoarthritis (OA) the cartilage appears macroscopically intact. However, this study demonstrates that the collagen network already changes in early OA by collagen fiber thinning and the formation of fibrocartilage-like tissue. Both nanoscopic deficiencies already occur in macroscopically intact regions of the human knee joint and are likely connected to processes that result in a weakened extracellular matrix. This study enhances the understanding of earliest progressive cartilage degeneration in the absence of external damage. The results suggest a determination of the mean collagen fiber thickness as a new target for the detection of early OA and a regulation of type I collagen synthesis as a new path for OA treatment.

Z3 Charge Density Wave of Silicon Atomic Chains on a Vicinal Silicon Surface.

An ideal one-dimensional electronic system is formed along atomic chains on Au-decorated vicinal silicon surfaces, but the nature of its low-temperature phases has been puzzling for last two decades. Here, we unambiguously identify the low-temperature structural distortion of this surface using high-resolution atomic force microscopy and scanning tunneling microscopy. The most important structural ingredient of this surface, the step-edge Si chains, are found to be strongly buckled, every third atom down, forming trimer unit cells. This observation is consistent with the recent model of rehybridized dangling bonds and rules out the antiferromagnetic spin ordering proposed earlier. The spectroscopy and electronic structure calculation indicate a charge density wave insulator with a Z3 topology, making it possible to exploit topological phases and excitations. The tunneling current was found to substantially lower the energy barrier between three degenerate CDW states, which induces a dynamically fluctuating CDW at very low temperature.