High-speed Atomic Force Microscopy Research Articles

Interfacial Layer Breaker: A Violation of Stokes’ Law in High-Speed Atomic Force Microscope Flows

Structured water near surfaces is important in nonclassical crystallization, biomineralization, and restructuring of cellular membranes. In addition to equilibrium structures, studied by atomic force microscopy (AFM), high-speed AFM (H-S AFM) can now detect piconewton forces in microseconds. With increasing speeds and decreasing tip diameters, there is a danger that continuum water models will not hold, and molecular dynamic (MD) simulations would be needed for accurate predictions. MD simulations, however, can only evolve over tens of nanoseconds due to memory and computational efficiency/speed limitations, so new methods are needed to bridge the gap. Here, we report a hybrid, multiscale simulation method, which can bridge the size and time scale gaps to existing experiments. Structured water is studied between a moving silica AFM colloidal tip and a cleaved mica surface. The computational domain includes 1,472,766 atoms. To mimic the effect of long-range hydrodynamic forces occurring in water, when moving the AFM tip at speeds from 5 × 10-7 to 30 m/s, a hybrid multiscale method with local atomistic resolution is used, which serves as an effective open-domain boundary condition. The multiscale simulation is thus equivalent to using a macroscopically large computational domain with equilibrium boundary conditions. Quantification of the drag force shows the breaking of continuum behavior. Nonmonotonic dependence on both the tip speed and distance from the surface implies breaking of the hydration layer around the moving tip at time scales smaller than water cluster formation and strong water compressibility effects at the highest speeds.

Effect of Alternative Developer Solutions on EUVL Patterning

This work explores the application of alternative developer solutions (“developers”) with the aim of understanding their potential effectivity in the reduction of resist-based stochastic pattern defects (or “stochastic defects”) in extreme ultraviolet lithography (EUVL). Specifically, the application of a quaternary ammonium hydroxide type aqueous developer; ethyltrimethylammonium hydroxide (ETMAH) in comparison to the industry de facto standard aqueous alkali tetramethylammonium hydroxide (TMAH) developer was investigated. Focusing on EUV exposed contact hole (CH) patterns on a typical chemically amplified resist (CAR), the effect of these developers on stochastic defects were assessed. As a result, patterning investigations showed that the lithographic performance of the CAR developed in ETMAH is comparable to the those obtained with TMAH. In situ resist dissolution analysis using the high-speed atomic force microscope (HS-AFM) confirms this as it showed that the rate of CH formation during resist dissolution in both developers are relatively the same. Moreover, it was also understood that compared to the commonly used alkali developer concentration of 0.26N, a lower ETMAH developer concentration of 0.20N resulted in stochastic defect margin improvement, while maintaining lithographic performance. In situ resist dissolution analysis showed an obvious slowing down of CH pattern formation rate at 0.20N concentration, suggesting the possibility that of over-dissolution at the higher concentration condition, translating to an increase in merging CH defects. The results from this study show the advantages of further pursuing optimal developers for EUVL. This is especially significant as these findings indicate how optimal developers mitigate resist-based stochastic defects while maintaining lithographic performance.

Functional Implications of Dynamic Structures of Intrinsically Disordered Proteins Revealed by High-Speed AFM Imaging

The unique functions of intrinsically disordered proteins (IDPs) depend on their dynamic protean structure that often eludes analysis. High-speed atomic force microscopy (HS-AFM) can conduct this difficult analysis by directly visualizing individual IDP molecules in dynamic motion at sub-molecular resolution. After brief descriptions of the microscopy technique, this review first shows that the intermittent tip-sample contact does not alter the dynamic structure of IDPs and then describes how the number of amino acids contained in a fully disordered region can be estimated from its HS-AFM images. Next, the functional relevance of a dumbbell-like structure that has often been observed on IDPs is discussed. Finally, the dynamic structural information of two measles virus IDPs acquired from their HS-AFM and NMR analyses is described together with its functional implications.

Membrane-mediated protein interactions drive membrane protein organization

The plasma membrane’s main constituents, i.e., phospholipids and membrane proteins, are known to be organized in lipid-protein functional domains and supercomplexes. No active membrane-intrinsic process is known to establish membrane organization. Thus, the interplay of thermal fluctuations and the biophysical determinants of membrane-mediated protein interactions must be considered to understand membrane protein organization. Here, we used high-speed atomic force microscopy and kinetic and membrane elastic theory to investigate the behavior of a model membrane protein in oligomerization and assembly in controlled lipid environments. We find that membrane hydrophobic mismatch modulates oligomerization and assembly energetics, and 2D organization. Our experimental and theoretical frameworks reveal how membrane organization can emerge from Brownian diffusion and a minimal set of physical properties of the membrane constituents.

Clarification of Surface Deswelling of Thermoresponsive Microgels by Electrophoresis.

Although many investigations of thermoresponsive microgels have been reported, their surface properties, which are crucial in colloid science, are still not fully understood. In this study, microgels with surface-localized charged groups were synthesized by precipitation polymerization, and their electrophoretic behaviors were analyzed using a modified version of Ohshima's equation to obtain two surface properties of the soft particles: the softness parameter and the surface charge density. This systematic evaluation allows us to discuss the thermoresponsiveness of the overall microgels and their surfaces separately. Furthermore, the validity of the surface properties obtained from electrophoresis was verified by comparing them with the results of seeded emulsion polymerization in the presence of the microgels and the force-indentation curves obtained via high-speed atomic force microscopy (HS-AFM).

Visualizing Intramolecular Dynamics of Membrane Proteins

Membrane proteins play important roles in biological functions, with accompanying allosteric structure changes. Understanding intramolecular dynamics helps elucidate catalytic mechanisms and develop new drugs. In contrast to the various technologies for structural analysis, methods for analyzing intramolecular dynamics are limited. Single-molecule measurements using optical microscopy have been widely used for kinetic analysis. Recently, improvements in detectors and image analysis technology have made it possible to use single-molecule determination methods using X-rays and electron beams, such as diffracted X-ray tracking (DXT), X-ray free electron laser (XFEL) imaging, and cryo-electron microscopy (cryo-EM). High-speed atomic force microscopy (HS-AFM) is a scanning probe microscope that can capture the structural dynamics of biomolecules in real time at the single-molecule level. Time-resolved techniques also facilitate an understanding of real-time intramolecular processes during chemical reactions. In this review, recent advances in membrane protein dynamics visualization techniques were presented.

Physical Properties and Shifting of the Extracellular Membrane Vesicles Attached to Living Bacterial Cell Surfaces.

Bacterial cells release nanometer-sized extracellular membrane vesicles (MVs) to deliver cargo molecules for use in mediating various biological processes. However, the detailed processes of transporting these cargos from MVs to recipient cells remain unclear because of the lack of imaging techniques to image nanometer-sized fragile vesicles in a living bacterial cell surface. Herein, we quantitatively demonstrated that the direct binding of MV to the cell surface significantly promotes hydrophobic quorum-sensing signal (C16-HSL) transportation to the recipient cells. Moreover, we analyzed the MV-binding process in the Paracoccus denitrificans cell surface using high-speed atomic force microscopy phase imaging. Although MV shapes were unaltered after binding to the cell surface, the physical properties of a group of single MV particles were shifted. Additionally, the phase shift values of MVs were higher than that of the cell's surfaces upon binding, whereas the phase shift values of the group of MVs were decreased during observation. The shifting physical properties occurred irreversibly only once for each MV during the observations. The decreasing phase shift values indicated alterations of chemical components in the MVs as well, thereby suggesting the dynamic process in which single MV particles deliver their hydrophobic cargo into the recipient cell. IMPORTANCE Compared to the increasing knowledge about MV release mechanisms from donor cells, the mechanism by which recipient cells receive cargo from MVs remains unknown. Herein, we have successfully imaged single MV-binding processes in living bacterial cell surfaces. Accordingly, we confirmed the shift in the MV hydrophobic properties after landing on the cell surface. Our results showed the detailed states and the attaching process of a single MV into the cell surface and can aid the development of a new model for MV reception into Gram-negative bacterial cell surfaces. The insight provided by this study is significant for understanding MV-mediated cell-cell communication mechanisms. Moreover, the AFM technique presented for nanometer-scaled mapping of dynamic physical properties alteration on a living cell could be applied for the analyses of various biological phenomena occurring on the cell surface, and it gives us a new view into the understanding of the phenotypes of the bacterial cell surface.

Structural and dynamic basis of DNA capture and translocation by mitochondrial Twinkle helicase.

Twinkle is a mitochondrial replicative helicase which can self-load onto and unwind mitochondrial DNA. Nearly 60 mutations on Twinkle have been linked to human mitochondrial diseases. Using cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM), we obtained the atomic-resolution structure of a vertebrate Twinkle homolog with DNA and captured in real-time how Twinkle is self-loaded onto DNA. Our data highlight the important role of the non-catalytic N-terminal domain of Twinkle. The N-terminal domain directly contacts the C-terminal helicase domain, and the contact interface is a hotspot for disease-related mutations. Mutations at the interface destabilize Twinkle hexamer and reduce helicase activity. With HS-AFM, we observed that a highly dynamic Twinkle domain, which is likely to be the N-terminal domain, can protrude ∼5 nm to transiently capture nearby DNA and initialize Twinkle loading onto DNA. Moreover, structural analysis and subunit doping experiments suggest that Twinkle hydrolyzes ATP stochastically, which is distinct from related helicases from bacteriophages.

Guide to studying intrinsically disordered proteins by high-speed atomic force microscopy.

Intrinsically disordered proteins (IDPs) are partially or entirely disordered. Their intrinsically disordered regions (IDRs) dynamically explore a wide range of structural space by their highly flexible nature. Due to this distinct feature largely different from structured proteins, conventional structural analyses relying on ensemble averaging is unsuitable for characterizing the dynamic structure of IDPs. Therefore, single-molecule measurement tools have been desired in IDP studies. High-speed atomic force microscopy (HS-AFM) is a unique tool that allows us to directly visualize single biomolecules at 2-3nm lateral and∼0.1nm vertical spatial resolution, and at sub-100ms temporal resolution under near physiological conditions, without any chemical labeling. HS-AFM has been successfully used not only to characterize the shape and motion of IDP molecules but also to visualize their function-related dynamics. In this article, after reviewing the principle and current performances of HS-AFM, we describe experimental considerations in the HS-AFM imaging of IDPs and methods to quantify molecular features from captured images. Finally, we outline recent HS-AFM imaging studies of IDPs.

Tip-scan high-speed atomic force microscopy with a uniaxial substrate stretching device for studying dynamics of biomolecules under mechanical stress.

High-speed atomic force microscopy (HS-AFM) is a powerful tool for studying the dynamics of biomolecules in vitro because of its high temporal and spatial resolution. However, multi-functionalization, such as combination with complementary measurement methods, environment control, and large-scale mechanical manipulation of samples, is still a complex endeavor due to the inherent design and the compact sample scanning stage. Emerging tip-scan HS-AFM overcame this design hindrance and opened a door for additional functionalities. In this study, we designed a motor-driven stretching device to manipulate elastic substrates for HS-AFM imaging of biomolecules under controllable mechanical stimulation. To demonstrate the applicability of the substrate stretching device, we observed a microtubule buckling by straining the substrate and actin filaments linked by α-actinin on a curved surface. In addition, a BAR domain protein BIN1 that senses substrate curvature was observed while dynamically controlling the surface curvature. Our results clearly prove that large-scale mechanical manipulation can be coupled with nanometer-scale imaging to observe biophysical effects otherwise obscured.

Spatiotemporal tracking of small extracellular vesicle nanotopology in response to physicochemical stresses revealed by HS-AFM.

Small extracellular vesicles (sEVs) play a crucial role in local and distant cell communication. The intrinsic properties of sEVs make them compatible biomaterials for drug delivery, vaccines, and theranostic nanoparticles. Although sEV proteomics have been robustly studied, a direct instantaneous assessment of sEV structure dynamics remains difficult. Here, we use the high-speed atomic force microscopy (HS-AFM) to evaluate nanotopological changes of sEVs with respect to different physicochemical stresses including thermal stress, pH, and osmotic stress. The sEV structure is severely altered at high-temperature, high-pH, or hypertonic conditions. Surprisingly, the spherical shape of the sEVs is maintained in acidic or hypotonic environments. Real-time observation by HS-AFM imaging reveals an irreversible structural change in the sEVs during transition of pH or osmolarity. HS-AFM imaging provides both qualitative and quantitative data at high spatiotemporal resolution (nanoscopic and millisecond levels). In summary, our study demonstrates the feasibility of HS-AFM for structural characterization and assessment of nanoparticles.

Genome editing technology and applications with the type I CRISPR system

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which are representative genome editing technologies, are classified into class 1 and class 2 in terms of evolutionary biology and are further classified into several subtypes. Class 2 CRISPR systems, including type II Cas9 and type V Cas12a, are the most commonly used for genome editing in eukaryotic cells, while type I CRISPR systems within Class 1 are also becoming available. Type I CRISPR recognizes longer target sequences than CRISPR-Cas9 and can induce large deletion mutations of several kilobases. These features demonstrate its potential as a novel and unique genome editing tool that can induce genetic disruption safely and reliably. Thus, it is expected to be utilized for gene therapy and industrial applications. Recently, the DNA cleavage mechanism of type I CRISPR has also revealed details from protein-complex analyses with X-ray crystallography, cryo-electron microscopy, and high-speed atomic force microscopy. The single-strand DNA trans-cleavage activity of type I CRISPR, called collateral activity, has broadened the potential application for CRISPR diagnostics, especially in the development of point-of-care testing methods for COVID-19. In this review, we present an overview of the type I CRISPR system, its application to genome editing, and genetic diagnosis using CRISPR-Cas3.

Actin-binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II.

We report a case in which sub-stoichiometric binding of an actin-binding protein has profound structural and functional consequences, providing an insight into the fundamental properties of actin regulation. Rng2 is an IQGAP contained in contractile rings in the fission yeast Schizosaccharomyces pombe Here, we used high-speed atomic force microscopy and electron microscopy and found that sub-stoichiometric binding of the calponin-homology actin-binding domain of Rng2 (Rng2CHD) induces global structural changes in skeletal muscle actin filaments, including shortening of the filament helical pitch. Sub-stoichiometric binding of Rng2CHD also reduced the affinity between actin filaments and muscle myosin II carrying ADP and strongly inhibited the motility of actin filaments on myosin II in vitro. On skeletal muscle myosin II-coated surfaces, Rng2CHD stopped the actin movements at a binding ratio of 11%. Rng2CHD also inhibited actin movements on myosin II of the amoeba Dictyostelium, but in this case, by detaching actin filaments from myosin II-coated surfaces. Thus, sparsely bound Rng2CHD induces apparently cooperative structural changes in actin filaments and inhibits force generation by actomyosin II.

A molecularly engineered, broad-spectrum anti-coronavirus lectin inhibits SARS-CoV-2 and MERS-CoV infection in vivo

Summary“Pan-coronavirus” antivirals targeting conserved viral components can be designed. Here, we show that the rationally engineered H84T-banana lectin (H84T-BanLec), which specifically recognizes high mannose found on viral proteins but seldom on healthy human cells, potently inhibits Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (including Omicron), and other human-pathogenic coronaviruses at nanomolar concentrations. H84T-BanLec protects against MERS-CoV and SARS-CoV-2 infection in vivo. Importantly, intranasally and intraperitoneally administered H84T-BanLec are comparably effective. Mechanistic assays show that H84T-BanLec targets virus entry. High-speed atomic force microscopy depicts real-time multimolecular associations of H84T-BanLec dimers with the SARS-CoV-2 spike trimer. Single-molecule force spectroscopy demonstrates binding of H84T-BanLec to multiple SARS-CoV-2 spike mannose sites with high affinity and that H84T-BanLec competes with SARS-CoV-2 spike for binding to cellular ACE2. Modeling experiments identify distinct high-mannose glycans in spike recognized by H84T-BanLec. The multiple H84T-BanLec binding sites on spike likely account for the drug compound’s broad-spectrum antiviral activity and the lack of resistant mutants.

Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies.

Atomic Force Microscopy (AFM) is widely used for topographic imaging of DNA and protein-DNA complexes in ambient conditions with nanometer resolution. In AFM studies of protein-DNA complexes, identifying the protein’s location on the DNA substrate is one of the major goals. Such studies require distinguishing between the DNA ends, which can be accomplished by end-specific labeling of the DNA substrate. We selected as labels three-way DNA junctions (3WJ) assembled from synthetic DNA oligonucleotides with two arms of 39–40 bp each. The third arm has a three-nucleotide overhang, GCT, which is paired with the sticky end of the DNA substrate generated by the SapI enzyme. Ligation of the 3WJ results in the formation of a Y-type structure at the end of the linear DNA mole cule, which is routinely identified in the AFM images. The yield of labeling is 69%. The relative orientation of arms in the Y-end varies, such dynamics were directly visualized with time-lapse AFM studies using high-speed AFM (HS-AFM). This labeling approach was applied to the characterization of the nucleosome arrays assembled on different DNA templates. HS-AFM experiments revealed a high dynamic of nucleosomes resulting in a spontaneous unraveling followed by disassembly of nucleosomes.

High-speed AFM reveals the dynamics of virus budding

Retrovirus assembly is coordinated by the viral polyprotein Gag, and after assembly, the particles are released from the host cell in a process called budding. Assembly of one of the best-studied retroviruses, human immunodeficiency virus-1 (HIV-1), occurs at the plasma membrane of infected cells (Fig. 1). Driven by Gag, an almost complete viral shell is formed when the particle buds from the cell via membrane fission (2). As other retroviruses, HIV-1 hijacks the host endosomal complex required for transport (ESCRT) machinery for budding.

Hydroxide films on mica form charge-stabilized microphases that circumvent nucleation barriers.

Crystal nucleation is facilitated by transient, nanoscale fluctuations that are extraordinarily difficult to observe. Here, we use high-speed atomic force microscopy to directly observe the growth of an aluminum hydroxide film from an aqueous solution and characterize the dynamically fluctuating nanostructures that precede its formation. Nanoscale cluster distributions and fluctuation dynamics show many similarities to the predictions of classical nucleation theory, but the cluster energy landscape deviates from classical expectations. Kinetic Monte Carlo simulations show that these deviations can arise from electrostatic interactions between the clusters and the underlying substrate, which drive microphase separation to create a nanostructured surface phase. This phase can evolve seamlessly from a low-coverage state of fluctuating clusters into a high-coverage nanostructured network, allowing the film to grow without having to overcome classical nucleation barriers.

High-speed Atomic Force Microscope Technology: A Review

:The atomic force microscope (AFM) is widely used in many fields such as biology, materials, and physics due to its advantages of simple sample preparation, high-resolution topography measurement and wide range of applications. However, the low scanning speed of traditional AFM limits its dynamics process monitoring and other further application. Therefore, the improvement of AFM scanning speed has become more and more important. In this review, the working principle of AFM is first proposed. Then, we introduce the improvements of cantilever, drive mechanism, and control method of the high-speed atomic force microscope (HS-AFM). Finally, we provide the next developments of HS-AFM.

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.

Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific single-stranded DNA (ssDNA) cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target double-stranded DNA (dsDNA) substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.