Molecular Scale Imaging Research Articles

Visualizing PIEZO1 Localization and Activity in hiPSC-Derived Single Cells and Organoids with HaloTag Technology.

PIEZO1 is critical to numerous physiological processes, transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of visualizing endogenous PIEZO1 activity and localization to understand its functional roles. To enable physiologically and clinically relevant studies on human PIEZO1, we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with advanced imaging, our chemogenetic platform allows precise visualization of PIEZO1 localization dynamics in various cell types. Furthermore, the PIEZO1-HaloTag hiPSC technology facilitates the non-invasive monitoring of channel activity across diverse cell types using Ca 2+ -sensitive HaloTag ligands, achieving temporal resolution approaching that of patch clamp electrophysiology. Finally, we used lightsheet imaging of hiPSC-derived neural organoids to achieve molecular scale imaging of PIEZO1 in three-dimensional tissue organoids. Our advances offer a novel platform for studying PIEZO1 mechanotransduction in human cells and tissues, with potential for elucidating disease mechanisms and targeted therapeutic development.

Molecular-Scale Imaging Enables Direct Visualization of Molecular Defects and Chain Structure of Conjugated Polymers.

Conjugated polymers have become materials of choice for applications ranging from flexible optoelectronics to neuromorphic computing, but their polydispersity and tendency to aggregate pose severe challenges to their precise characterization. Here, the combination of vacuum electrospray deposition (ESD) with scanning tunneling microscopy (STM) is used to acquire, within the same experiment, assembly patterns, full mass distributions, exact sequencing, and quantification of polymerization defects. In a first step, the ESD-STM results are successfully benchmarked against NMR for low molecular mass polymers, where this technique is still applicable. Then, it is shown that ESD-STM is capable of reaching beyond its limits by characterizing, with the same accuracy, samples that are inaccessible to NMR. Finally, a recalibration procedure is proposed for size exclusion chromatography (SEC) mass distributions, using ESD-STM results as a reference. The distinctiveness of the molecular-scale information obtained by ESD-STM highlights its role as a crucial technique for the characterization of conjugated polymers.

Direct Observation of Solvent–Reaction Intermediate Interactions in Heterogeneously Catalyzed Alcohol Coupling

The relative stability of reactive intermediates and reactants on a surface, which dictates the rate and selectivity of catalytic reactions in both gas and liquid phases, is dependent on numerous factors. One well-established example is secondary interactions, such as van der Waals interactions between the catalyst surface and the pendant group of the intermediate, which can govern reaction selectivity for coupling reactions. Herein, we directly show that interactions between adsorbed reaction intermediates and reactant molecules increase the binding energy and affects the geometrical arrangement of coadsorbed reactant/solvent molecules. Evidence for this effect is demonstrated for the oxidative coupling reaction of methanol on a single crystal gold (Au(110)) surface. The rate-limiting reaction intermediate for methanol self-coupling, methoxy, stabilizes excess adsorbed methanol, which desorbs as a result of beta-hydride decomposition of the adsorbed methoxy. Direct molecular-scale imaging by scanning tunneling microscopy, supplemented by density functional theory, revealed interactive structures formed by methoxy and coadsorbed methanol. Interactions between the methoxy intermediate and coadsorbed methanol stabilizes a hydrogen-bonded network comprising methoxy and methanol by a minimum of 0.13 eV per methanol molecule. Inclusion of such interactions between reaction intermediates and coadsorbed reactants and solvents in kinetic models is important for microkinetic analysis of the rates and selectivities of catalytic reactions in both the gas and liquid phases whenever appreciable coverages of species from the ambient phase exist.

Label-retention expansion microscopy.

Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

Combined AFM and super-resolution localisation microscopy: Investigating the structure and dynamics of podosomes

Podosomes are mechanosensitive attachment/invasion structures that form on the matrix-adhesion interface of cells and protrude into the extracellular matrix to probe and remodel. Despite their central role in many cellular processes, their exact molecular structure and function remain only partially understood. We review recent progress in molecular scale imaging of podosome architecture, including our newly developed localisation microscopy technique termed HAWK which enables artefact-free live-cell super-resolution microscopy of podosome ring proteins, and report new results on combining fluorescence localisation microscopy (STORM/PALM) and atomic force microscopy (AFM) on one setup, where localisation microscopy provides the location and dynamics of fluorescently labelled podosome components, while the spatial variation of stiffness is mapped with AFM. For two-colour localisation microscopy we combine iFluor-647, which has previously been shown to eliminate the need to change buffer between imaging modes, with the photoswitchable protein mEOS3.2, which also enables live cell imaging.

Real-Time Molecular-Scale Imaging of Dynamic Network Switching between Covalent Organic Frameworks.

The in situ on-surface conversion process from boroxine-linked covalent organic frameworks (COFs) to boronate ester-linked COFs is triggered and catalyzed at room temperature by an electric field and monitored with scanning tunneling microscopy (STM). The adaptive behavior within the generated dynamic covalent libraries (DCLs) was revealed, providing in-depth understanding of the dynamic network switching process.

Applications of Label-Retention Expansion Microscopy on Biological Systems at Various Scales

Expansion Microscopy (ExM) provides super resolution by physically expanding biological samples in swellable polymer. Fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. To overcome this bottleneck, we developed Label-Retention Expansion Microscopy (LR-ExM) and a set of novel trifunctional chemical linkers that prevent fluorescence loss and enhance the labeling efficiency. With LR-ExM, the currently achieved post-expansion resolutions of ∼70 nm with confocal microscopy, ∼30 nm with SIM, and ∼5 nm with STORM are suitable to cover a wide range of biological questions at various scales. In this work, we reported structural studies of lamina network using confocal LR-ExM, symmetry of cilia using LR-ExSIM, and polyhedral lattice of clathrin-coated pits in situ using LR-ExSTORM. This method robustly provides molecular-scale resolution in cells and tissues.

Real Space Demonstration of Induced Crystalline 3D Nanostructuration of Organic Layers.

The controlled 3D nanostructuration of molecular layers of the semiconducting molecules C22H14 (pentacene) and N,N'-dioctyl-3,4,9,10-perylene tetracarboxylic diimide (PTCDI-C8) is addressed. A tip-assisted method using atomic force microscopy (AFM) is developed for removing part of the organic material and relocating it in up to six layer thick nanostructures. Moreover, unconventional molecular scale imaging combining diverse friction force microscopy modes reveals the stacking sequence of the piled layers. In particular, we unambiguously achieve epitaxial growth, an issue of fundamental importance in thin film strategies for the nanostructuration of more efficient organic nanodevices.

Monoclonal Antibody Aggregation Intermediates Visualized by Atomic Force Microscopy

Ubiquitous but highly variable processes of therapeutic protein aggregation remain poorly characterized, especially in the context of common infusion reactions and clinical immunogenicity. Among the numerous challenges is the characterization of intermediate steps that lead to the appearance of precipitates. Although the biophysical methods for elucidation of secondary and tertiary structures as well as overall size distribution are typically well established in the development laboratories, the use of molecular scale imaging techniques is still relatively rare due to low throughput and technical complexity. In this work, we present the use of atomic force microscopy to examine morphology of monoclonal antibody aggregates. Despite varying in primary structure as a result of different complementarity defining regions, most antibodies studied exhibited a similar aggregation intermediate consisting of several monomers. However, the manner of subsequent condensation of these oligomers appeared to differ between the antibodies, suggesting stability-dependent mechanisms.

1P142 1E1240 脂質膜/水界面の3次元走査型原子間力顕微鏡による分子スケールイメージング(水・水和/電解質,口頭発表,第48回日本生物物理学会年会)

1P142 1E1240 脂質膜/水界面の3次元走査型原子間力顕微鏡による分子スケールイメージング(水・水和/電解質,口頭発表,第48回日本生物物理学会年会)

3P-121 周波数変調原子間力顕微鏡によるモデル生体膜上に形成された水和層の分子分解能観察(水・水和/電解質,日本生物物理学会若手奨励賞選考会,若手招待講演,第46回日本生物物理学会年会)

3P-121 周波数変調原子間力顕微鏡によるモデル生体膜上に形成された水和層の分子分解能観察(水・水和/電解質,日本生物物理学会若手奨励賞選考会,若手招待講演,第46回日本生物物理学会年会)

Molecular Scale Imaging of F-Actin Assemblies Immobilized on a Photopolymer Surface

A photo-immobilization based process is presented for direct imaging of hierarchical assemblies of biopolymers using atomic force microscopy (AFM). The technique was used to investigate the phase behavior of F-actin aggregates as a function of concentration of the divalent cation Mg2+. The data provided direct experimental evidence of a coil-on-coil (braided) structure of F-actin bundles formed at high Mg2+ concentrations. At intermediate Mg2+ concentrations, the data showed the first images of the two-dimensional nematic rafts discovered by recent x-ray studies and theoretical treatments.

Molecular-scale imaging of unstained deoxyribonucleic acid fibers by phase transmission electron microscopy

The molecular structure of deoxyribonucleic acid (DNA) fibers was observed by a phase reconstruction method called three-dimensional Fourier filtering using a 200kV transmission electron microscope. The characteristic helical structure and the spacing of adjacent base pairs of DNA were partially resolved due to an improved signal-to-noise ratio and resolution enhancement by the phase reconstruction although the molecular structure was damaged by the electron beam irradiation. In the spherical aberration-free phase images, the arrangements of single atom-sized spots forming sinusoidal curves were sometimes observed, which seem to be the contrast originating in the sulfur atoms along the main chains.

True Molecular-Scale Imaging in Atomic Force Microscopy: Experiment and Modeling

Imaging with molecular-resolution achieved in tapping mode atomic force microscopy (AFM) opened a number of questions concerning image contrast and its variations. These issues are addressed in the interplay between experiment and theory in studies of polydiacetylene crystal. The dependence of image features on tip force and tip size has been revealed in theoretical simulations where bifurcation phenomenon played a major role. Experimental observations of periodical surface structures with molecular-scale single defects do not necessary prove true molecular-scale resolution because a number of periodical features seen in AFM images can originate in a result of bifurcation.

Molecular Scale Imaging with a Multilayer Superlens

AbstractIt has been experimentally demonstrated that a single layer of silver functions as a “superlens” [Fang et al, Science 308, 534 (2005)], providing image resolution much better than the diffraction limit. Resolution as high as 60 nanometer (λ/6) half-pitch was achieved. In this paper, we explore the possibility of further refining the image resolution using a “multilayer superlens” design. With optimized design of silver-alumina multilayer superlens, our numerical simulations show a feasibility of resolving 15nm features, about 1/26th of the illumination wavelength. We present preliminary experimental results targeted towards achieving the molecular scale imaging resolution. The development of potential low-loss and high resolution superlens opens the door to exciting applications in nanoscale optical metrology and nanomanufacturing.

Three Dimensional Molecular Scale Imaging of Plasma Proteins Under Aqueous Conditions

Atomic force microscopy (AFM) provides unique opportunities to study cell-surface and molecular scale interactions in three dimensions under aqueous conditions. In AFM, a small probe attached to cantilever (fig. 1) is used for interacting with a sample to obtain sensitive measurements of molecular structure, intermolecular forces (sub-nN) and interfacial properties. The data ouput can be presented in the form of a topographical image, force map or viscoelastic response (e.g., Figs. 2-4). Thus, AFM combines high resolution imaging with the ability to measure surface-dependent intermolecular forces and properties. AFM also is amenable for coupling with optical imaging methods including immunofluoresence and gold bead labeling.This presentation will focus primarily on the use of AFM for molecular level imaging of plasma proteins, von Willebrand Factor (vWF) and fibrinogen. Both proteins play central roles in the regulation of hemostasis and thrombosis by participating in coagulation or by facilitating adhesion, spreading and aggregation of activated platelets.

Burgeoning markets for STM

Ten years (and one Nobel Prize) after Gerd Binnig and Heinrich Rohrer invented it, the scanning tunnelling microscope's potential for atomic- and molecular-scale imaging of surface structure, coupled with its ability to map the topography of objects with lateral dimensions in the tens of μm range, is being exploited in a diverse array of research disciplines. Surface science (defect analysis, nucleation and growth processes, chemical reactivity and adsorption), biological (biomolecular structures, binding of pharmaceutical drugs to DNA and protein molecules) and technological applications (friction studies at an atomic level, nano-machining) are just a few of the areas where the STM is proving itself an invaluable research tool.

Molecular resolution of Langmuir-Blodgett monolayers on tungsten diselenide by scanning tunneling microscopy

Langmuir-Blodgett (LB) monolayers of 22-tricosenoic acid deposited on layered tungsten diselenide (WSe2) have been investigated by Scanning Tunneling Microscopy (STM) in air. Reproducible molecular scale imaging of extended areas (16×16 nm) of the films was possible over more than two months after preparation. This indicates a high stability of the films on this type of polar substrates. The two-dimensional lattice of the organic film is incommensurate to the lattice of the WSe2 substrates. The STM experiments also indicate that the tip partially penetrates the organic film without destroying the film structure.

Molecular-Scale Imaging of Clay Mineral Surfaces with the Atomic Force Microscope

AbstractSpecimen samples of Crook County montmorillonite and Silver Hill illite, purified and prepared in the Na-form, were imaged under 80% relative humidity using an atomic force microscope. The direct images showed clearly the hexagonal array of hexagonal rings of oxygen ions expected for the basal planes of 2:1 phyllosilicates. Fourier transformation of the digital information obtained by the microscope scanning tip led to an estimate of 5.1 ± 0.3 Å for the nearest-neighbor separation, in agreement with the ideal nearest-neighbor spacing of 5.4 Å for hexagonal rings as derived from X-ray powder diffraction data. The atomic force microscope should prove to be a useful tool for the molecular-scale resolution of clay mineral surfaces that contain adsorbed macromolecules.