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1.
Rev Sci Instrum ; 90(11): 113701, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31779413

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.

2.
Sci Rep ; 9(1): 7218, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31076614

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.

3.
Cell Rep ; 14(8): 1813-8, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26904946

RESUMO

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 ∼ 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 ∼ 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 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/metabolismo
4.
Small ; 11(8): 976-84, 2015 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-25336288

RESUMO

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 (∼25 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 , Tampões (Química) , Drosophila , Escherichia coli/metabolismo , Histonas/química , Íons , Nanotecnologia/métodos , Proteínas/química , Sais/química , Propriedades de Superfície
5.
ACS Nano ; 8(1): 35-43, 2014 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-24295288

RESUMO

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 , Permeabilidade
6.
Langmuir ; 29(4): 1206-10, 2013 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-23244532

RESUMO

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.

7.
Ultramicroscopy ; 111(12): 1659-69, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22094372

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.


Assuntos
Microscopia de Força Atômica/instrumentação , Microscopia de Força Atômica/métodos , Análise Espectral/métodos , Algoritmos , Calibragem , Ligantes , Modelos Teóricos , Análise Espectral/instrumentação , Eletricidade Estática
8.
Cell ; 147(5): 979-82, 2011 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-22118456

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/métodos
9.
Nano Lett ; 11(12): 5489-93, 2011 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-22017420

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 Ópticas
10.
Nano Lett ; 11(10): 4107-12, 2011 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-21848283

RESUMO

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.

11.
Nanotechnology ; 20(16): 165703, 2009 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-19420576

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.

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