RESUMO
Atomic force microscopy allows visualization of biomolecules with nanometer resolution under physiological conditions. Recent advances have improved the time resolution of the technique from minutes to tens of milliseconds, meaning that it is now possible to watch single biomolecules in action in real time. Here, we review this development.
Assuntos
Microscopia de Força Atômica/métodos , Nanopartículas/ultraestrutura , Bactérias/ultraestrutura , Células Eucarióticas/ultraestrutura , Nanopartículas/química , Nanotecnologia/métodosRESUMO
Nitrogen-vacancy (NV) magnetometry is a new technique for imaging spin waves in magnetic materials. It detects spin waves by their microwave magnetic stray fields, which decay evanescently on the scale of the spin-wavelength. Here, we use nanoscale control of a single-NV sensor as a wavelength filter to characterize frequency-degenerate spin waves excited by a microstrip in a thin-film magnetic insulator. With the NV probe in contact with the magnet, we observe an incoherent mixture of thermal and microwave-driven spin waves. By retracting the tip, we progressively suppress the small-wavelength modes until a single coherent mode emerges from the mixture. In-contact scans at low drive power surprisingly show occupation of the entire isofrequency contour of the two-dimensional spin-wave dispersion despite our one-dimensional microstrip geometry. Our distance-tunable filter sheds light on the spin-wave band occupation under microwave excitation and opens opportunities for imaging magnon condensates and other coherent spin-wave modes.
RESUMO
Controlling magnon densities in magnetic materials enables driving spin transport in magnonic devices. We demonstrate the creation of large, out-of-equilibrium magnon densities in a thin-film magnetic insulator via microwave excitation of coherent spin waves and subsequent multimagnon scattering. We image both the coherent spin waves and the resulting incoherent magnon gas using scanning-probe magnetometry based on electron spins in diamond. We find that the gas extends unidirectionally over hundreds of micrometers from the excitation stripline. Surprisingly, the gas density far exceeds that expected for a boson system following a Bose-Einstein distribution with a maximum value of the chemical potential. We characterize the momentum distribution of the gas by measuring the nanoscale spatial decay of the magnetic stray fields. Our results show that driving coherent spin waves leads to a strong out-of-equilibrium occupation of the spin-wave band, opening new possibilities for controlling spin transport and magnetic dynamics in target directions.
RESUMO
While graphene may appear to be the ultimate support membrane for transmission electron microscopy (TEM) imaging of DNA nanostructures, very little is known if it poses an advantage over conventional carbon supports in terms of resolution and contrast. Microscopic investigations are carried out on DNA origami nanoplates that are supported onto freestanding graphene, using advanced TEM techniques, including a new dark-field technique that is recently developed in our lab. TEM images of stained and unstained DNA origami are presented with high contrast on both graphene and amorphous carbon membranes. On graphene, the images of the origami plates show severe unwanted distortions, where the rectangular shape of the nanoplates is significantly distorted. From a number of comparative control experiments, it is demonstrated that neither staining agents, nor screening ions, nor the level of electron-beam irradiation cause this distortion. Instead, it is suggested that origami nanoplates are distorted due to hydrophobic interaction of the DNA bases with graphene upon adsorption of the DNA origami nanoplates.
Assuntos
DNA/química , Grafite/química , Microscopia Eletrônica de Transmissão , Nanoestruturas/química , Torção Mecânica , DNA/análise , DNA/ultraestrutura , Grafite/análise , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão/métodos , Microscopia Eletrônica de Transmissão/tendências , Nanocompostos/análise , Nanocompostos/química , Nanocompostos/ultraestrutura , Nanoestruturas/ultraestrutura , Nanotecnologia/métodos , Nanotecnologia/tendências , Conformação de Ácido NucleicoRESUMO
The accessibility of DNA is determined by the number, position, and stability of nucleosomes, complexes consisting of a core of 8 histone proteins with DNA wrapped around it. Since the structure and dynamics of nucleosomes affects essential cellular processes, they are the subject of many current studies. Here, high-speed atomic force microscopy is used to visualize dynamic processes in nucleosomes and tetrasomes (subnucleosomal structures that contain 4 rather than 8 histones in the protein core). Nucleosomes can spontaneously disassemble in a process (at a 1 second timescale). For tetrasomes, multiple dynamic phenomena are observed. For example, during disassembly the formation of a DNA loop (â¼25 nm in length) is seen, which remains stable for several minutes. For intact tetrasomes, dynamics in the form of sliding and reversible hopping between stable positions along the DNA are observed. The data emphasize that tetrasomes are not merely static objects but highly dynamic. Since tetrasomes (in contrast to nucleosomes) can stay on the DNA during transcription, the observed tetrasome dynamics is relevant for an understanding of the nucleosomal dynamics during transcription. These results illustrate the diversity of nucleosome dynamics and demonstrate the ability of high speed AFM to characterize protein-DNA interactions.
Assuntos
DNA/química , Microscopia de Força Atômica , Nucleossomos/química , Nucleossomos/ultraestrutura , Animais , Soluções Tampão , Drosophila , Escherichia coli/metabolismo , Histonas/química , Íons , Nanotecnologia/métodos , Proteínas/química , Sais/química , Propriedades de SuperfícieRESUMO
Cytoskeletal protein filaments such as actin and microtubules confer mechanical support to cells and facilitate many cellular functions such as motility and division. Recent years have witnessed the development of a variety of molecular scaffolds that mimic such filaments. Indeed, filaments that are programmable and compatible with biological systems may prove useful in studying or substituting such proteins. Here, we explore the use of ssRNA tiles to build and modify filaments in vitro. We engineer a number of functionalities that are crucial to the function of natural proteins filaments into the ssRNA tiles, including the abilities to assemble or disassemble filaments, to tune the filament stiffness, to induce membrane binding, and to bind proteins. This work paves the way for building dynamic cytoskeleton-mimicking systems made out of rationally designed ssRNA tiles that can be transcribed in natural or synthetic cells.
Assuntos
Citoesqueleto , Microtúbulos , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismoRESUMO
Using conducting probe atomic force microscopy (CAFM), we show that electrical conductivity in oligothiophene molecular films deposited on SiO(2)/Si wafers is extremely sensitive to degree of crystalline order in the film. By locally distorting the molecular order in the films through the controlled application of pressure with the AFM tip, the lateral charge transport was reduced by factors varying from 2 to 10, even when no changes in the height of the film could be observed.
RESUMO
Transmission electron microscopy was used to determine the structure of molecular films of self-assembled monolayers of pentathiophene derivatives supported on various electron transparent substrates. Despite the extreme beam sensitivity of the monolayers, structural crystallographic maps were obtained that revealed the nanoscale structure of the film. The image resolution is determined by the minimum beam diameter that the radiation hardness of the monolayer can support, which in our case is about 90 nm for a beam current of 5 × 10(6) e(-)/s. Electron diffraction patterns were collected while scanning a parallel electron beam over the film. These maps contain uncompromised information of the size, symmetry and orientation of the unit cell, orientation and structure of the domains, degree of crystallinity, and their variation on the micrometer scale, which are crucial to understand the electrical transport properties of the organic films. This information allowed us to track small changes in the unit cell size driven by the chemical modification of the support film.
Assuntos
Membranas Artificiais , Microscopia Eletrônica/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Compostos Orgânicos/química , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Using conducting probe atomic force microscopy (CAFM) we have investigated the electrical conduction properties of monolayer films of a pentathiophene derivative on a SiO(2)/Si-p+ substrate. By a combination of current-voltage spectroscopy and current imaging we show that lateral charge transport takes place in the plane of the monolayer via hole injection into the highest occupied molecular orbitals of the pentathiophene unit. Our CAFM data suggest that the conductivity is anisotropic relative to the crystalline directions of the molecular lattice.
RESUMO
Single-molecule force-spectroscopy methods such as magnetic and optical tweezers have emerged as powerful tools for the detailed study of biomechanical aspects of DNA-enzyme interactions. As typically only a single molecule of DNA is addressed in an individual experiment, these methods suffer from a low data throughput. Here, we report a novel method for targeted, nonrandom immobilization of DNA-tethered magnetic beads in regular arrays through microcontact printing of DNA end-binding labels. We show that the increase in density due to the arrangement of DNA-bead tethers in regular arrays can give rise to a one-order-of-magnitude improvement in data-throughput in magnetic tweezers experiments. We demonstrate the applicability of this technique in tweezers experiments where up to 450 beads are simultaneously tracked in parallel, yielding statistical data on the mechanics of DNA for 357 molecules from a single experimental run. Our technique paves the way for kilo-molecule force spectroscopy experiments, enabling the study of rare events in DNA-protein interactions and the acquisition of large statistical data sets from individual experimental runs.
Assuntos
DNA/química , Imãs/química , Microscopia de Força Atômica/métodos , Fenômenos Biomecânicos , Análise de Sequência com Séries de Oligonucleotídeos , Pinças ÓpticasRESUMO
Structural maintenance of chromosome (SMC) protein complexes are able to extrude DNA loops. While loop extrusion constitutes a fundamental building block of chromosomes, other factors may be equally important. Here, we show that yeast cohesin exhibits pronounced clustering on DNA, with all the hallmarks of biomolecular condensation. DNA-cohesin clusters exhibit liquid-like behavior, showing fusion of clusters, rapid fluorescence recovery after photobleaching and exchange of cohesin with the environment. Strikingly, the in vitro clustering is DNA length dependent, as cohesin forms clusters only on DNA exceeding 3 kilo-base pairs. We discuss how bridging-induced phase separation, a previously unobserved type of biological condensation, can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in yeast cells in vivo, a fraction of cohesin associates with chromatin in a manner consistent with bridging-induced phase separation. Biomolecular condensation by SMC proteins constitutes a new basic principle by which SMC complexes direct genome organization.
Assuntos
Proteínas Cromossômicas não Histona , Saccharomyces cerevisiae , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos , DNA/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
Structural maintenance of chromosome (SMC) protein complexes are the key organizers of the spatiotemporal structure of chromosomes. The condensin SMC complex has recently been shown to be a molecular motor that extrudes large loops of DNA, but the mechanism of this unique motor remains elusive. Using atomic force microscopy, we show that budding yeast condensin exhibits mainly open 'O' shapes and collapsed 'B' shapes, and it cycles dynamically between these two states over time, with ATP binding inducing the O to B transition. Condensin binds DNA via its globular domain and also via the hinge domain. We observe a single condensin complex at the stem of extruded DNA loops, where the neck size of the DNA loop correlates with the width of the condensin complex. The results are indicative of a type of scrunching model in which condensin extrudes DNA by a cyclic switching of its conformation between O and B shapes.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Cromossomos Fúngicos/ultraestrutura , DNA Fúngico/química , DNA Fúngico/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expressão Gênica , Microscopia de Força Atômica , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
A new method for extracting quantitative data from amplitude modulation dynamic force-distance measurements is developed. The method is based on the harmonic oscillator model of vibrating atomic force microscope cantilevers, and is capable of extracting both the conservative and dissipative parts of the tip-sample interaction from a measurement of oscillation amplitude and phase as a function of distance. Numerical simulations are used to demonstrate the validity of the method. Further proof of the accuracy of this method is provided by a measurement of electrostatic forces between an AFM tip and a graphite sample.
RESUMO
We have realized a microstrip based terahertz (THz) near field cantilever that enables quantitative measurements of the impedance of the probe tip at THz frequencies (0.3 THz). A key feature is the on-chip balanced hybrid coupler that serves as an interferometer for passive signal cancellation to increase the readout circuit sensitivity despite extreme impedance mismatch at the tip. We observe distinct changes in the reflection coefficient of the tip when brought into contact with different dielectric (Si, SrTiO3) and metallic samples (Au). By comparing finite element simulations, we determine the sensitivity of our THz probe to be well below 0.25 fF. The cantilever further allows for topography imaging in a conventional atomic force microscope mode. Our THz cantilever removes several critical technology challenges and thus enables a shielded cantilever based THz near field microscope.
RESUMO
Over the last few years, tremendous progress has been made in visualizing biologically important macromolecules using transmission electron microscopy (TEM) and understanding their structure-function relation. Yet, despite the importance of DNA in all forms of life, TEM visualization of individual DNA molecules in its native unlabeled form has remained extremely challenging. Here, we present high-contrast images of unstained single-layer DNA nanostructures that were obtained using advanced in-focus phase contrast TEM techniques. These include sub-Ångstrom low voltage electron microscopy (SALVE), the use of a volta-potential phase plate (VPP), and dark-field (DF) microscopy. We discuss the advantages and drawbacks of these techniques for broad applications in structural biology and materials science.
Assuntos
DNA/química , Microscopia Eletrônica de Transmissão/métodos , Nanoestruturas/química , Processamento de Imagem Assistida por Computador , Microscopia de Contraste de FaseRESUMO
Structural maintenance of chromosomes (SMC) protein complexes, including cohesin and condensin, play key roles in the regulation of higher-order chromosome organization. Even though SMC proteins are thought to mechanistically determine the function of the complexes, their native conformations and dynamics have remained unclear. Here, we probe the topology of Smc2-Smc4 dimers of the S. cerevisiae condensin complex with high-speed atomic force microscopy (AFM) in liquid. We show that the Smc2-Smc4 coiled coils are highly flexible polymers with a persistence length of only â¼ 4 nm. Moreover, we demonstrate that the SMC dimers can adopt various architectures that interconvert dynamically over time, and we find that the SMC head domains engage not only with each other, but also with the hinge domain situated at the other end of the â¼ 45-nm-long coiled coil. Our findings reveal structural properties that provide insights into the molecular mechanics of condensin complexes.
Assuntos
Proteínas de Transporte/química , Proteínas Cromossômicas não Histona/química , Cromossomos Fúngicos/química , Proteínas Nucleares/química , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/ultraestrutura , Expressão Gênica , Processamento de Imagem Assistida por Computador , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Imagem Molecular , Método de Monte Carlo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios Proteicos , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
While DNA origami is a popular and versatile platform, its structural properties are still poorly understood. In this study we use solid-state nanopores to investigate the ionic permeability and mechanical properties of DNA origami nanoplates. DNA origami nanoplates of various designs are docked onto solid-state nanopores where we subsequently measure their ionic conductance. The ionic permeability is found to be high for all origami nanoplates. We observe the conductance of docked nanoplates, relative to the bare nanopore conductance, to increase as a function of pore diameter, as well as to increase upon lowering the ionic strength. The honeycomb lattice nanoplate is found to have slightly better overall performance over other plate designs. After docking, we often observe spontaneous discrete jumps in the current, a process which can be attributed to mechanical buckling. All nanoplates show a nonlinear current-voltage dependence with a lower conductance at higher applied voltages, which we attribute to a physical bending deformation of the nanoplates under the applied force. At sufficiently high voltage (force), the nanoplates are strongly deformed and can be pulled through the nanopore. These data show that DNA origami nanoplates are typically very permeable to ions and exhibit a number of unexpected mechanical properties, which are interesting in their own right, but also need to be considered in the future design of DNA origami nanostructures.
Assuntos
DNA/química , Nanoestruturas , Íons , PermeabilidadeRESUMO
Reliable measurement of electrical charge transport in molecular layers is a delicate task that requires establishing contacts with electrodes without perturbing the molecular structure of the film. We show how this can be achieved by means of novel device consisting of ultra-flat electrodes separated by insulating material to support the molecular film. We show the fabrication process of these electrodes using a replica technique where gold electrodes are embedded in a silicon oxide film deposited on the angstrom-level flat surface of a silicon wafer. Importantly, the co-planarity of the electrode and oxide areas of the substrate was in the sub-nanometer range. We illustrate the capabilities of the system by mapping the distribution of electrical transport pathways in molecular thin films of self-assembled oligothiophene derivatives using conductive atomic force microscopy. In comparison with traditional bottom contact non-coplanar electrodes, the films deposited on our electrodes exhibited contact resistances lower by a factor of 40 than that of the similar but non-coplanar electrodes.
RESUMO
Single-molecule force spectroscopy studies performed by Atomic Force Microscopes (AFMs) strongly rely on accurately determined cantilever spring constants. Hence, to calibrate cantilevers, a reliable calibration protocol is essential. Although the thermal noise method and the direct Sader method are frequently used for cantilever calibration, there is no consensus on the optimal calibration of soft and V-shaped cantilevers, especially those used in force spectroscopy. Therefore, in this study we aimed at establishing a commonly accepted approach to accurately calibrate compliant and V-shaped cantilevers. In a round robin experiment involving eight different laboratories we compared the thermal noise and the Sader method on ten commercial and custom-built AFMs. We found that spring constants of both rectangular and V-shaped cantilevers can accurately be determined with both methods, although the Sader method proved to be superior. Furthermore, we observed that simultaneous application of both methods on an AFM proved an accurate consistency check of the instrument and thus provides optimal and highly reproducible calibration. To illustrate the importance of optimal calibration, we show that for biological force spectroscopy studies, an erroneously calibrated cantilever can significantly affect the derived (bio)physical parameters. Taken together, our findings demonstrated that with the pre-established protocol described reliable spring constants can be obtained for different types of cantilevers.