High-speed atomic force microscopy: Imaging and force spectroscopy

Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.     

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

Advances in microscopy have contributed to many biologic discoveries. Electron microscopic techniques such as cryo-electron tomography are remarkable tools for imaging the interiors of bacterial cells in the near-native state, whereas optical microscopic techniques such as fluorescence imaging are useful for following the dynamics of specific single molecules in living cells. Neither technique, however, can be used to visualize the structural dynamics of a single molecule at high resolution in living cells. In the present study, we used high-speed atomic force microscopy (HS-AFM) to image the molecular dynamics of living bacterial cell surfaces. HS-AFM visualizes the dynamic molecular processes of isolated proteins at sub-molecular resolution without the need for complicated sample preparation. In the present study, magnetotactic bacterial cells were anchored in liquid medium on substrate modified by poly-l-lysine and glutaraldehyde. High-resolution HS-AFM images of live cell surfaces showed that the bacterial outer membrane was covered with a net-like structure comprising holes and the hole rims framing them. Furthermore, HS-AFM captured the dynamic movement of the surface ultrastructure, showing that the holes in the net-like structure slowly diffused in the cell surface. Nano-dissection revealed that porin trimers constitute the net-like structure. Here, we report for the first time the direct observation of dynamic molecular architectures on a live cell surface using HS-AFM.     

High-speed atomic force microscopy and its future prospects

Various techniques have been developed and used to investigate how proteins produce complex biological architectures and phenomena. Among these techniques, high-speed atomic force microscopy (HS-AFM) holds a unique position. It is only HS-AFM that allows the simultaneous assessment of structure and dynamics of single protein molecules in action. This new microscopy tool has been successfully applied to a variety of proteins, from motor proteins to membrane proteins, antibodies, enzymes, and even to intrinsically disordered proteins. And yet there still remain many biomolecular phenomena that cannot be addressed by HS-AFM in its current form. Here, I present a brief history of HS-AFM development, describe the current state of HS-AFM, and then discuss which new biological scanning probe microscopy techniques will be coming up next.     

Software for drift compensation, particle tracking and particle analysis of high-speed atomic force microscopy image series

Atomic force microscopy (AFM) image acquisition is performed by raster-scanning a faint tip with respect to the sample by the use of a piezoelectric stage that is guided by a feedback system. This process implies that the resulting images feature particularities that distinguish them from images acquired by other techniques, such as the drift of the piezoelectric elements, the unequal image contrast along the fast- and the slow-scan axes, the physical contact between the tip of nondefinable geometry and the sample, and the feedback parameters. Recently, high-speed AFM (HS-AFM) has been introduced, which allows image acquisition about three orders of magnitude faster (500-100 ms frame rate) than conventional AFM (500 s to 100 s frame rate). HS-AFM produces image sequences, large data sets, which report biological sample dynamics. To analyze these movies, we have developed a software package that (i) adjusts individual scan lines and images to a common contrast and z-scale, (ii) filters specifically those scan lines where increased or insufficient force was applied, (iii) corrects for piezo-scanner drift, (iv) defines particle localization and angular orientation, and (v) performs particle tracking to analyze the lateral and rotation displacement of single molecules.     

High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies

BackgroundHigh-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy.MethodsScanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever.ResultsIn tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated.ConclusionsThis study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research.General significanceWe achieved an imaging rate of ~3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.     

Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy

High-speed atomic force microscopy (HS-AFM) allows direct visualization of dynamic structural changes and processes of functioning biological molecules in physiological solutions, at subsecond to sub-100-ms temporal and submolecular spatial resolution. Unlike fluorescence microscopy, wherein the subset of molecular events that you see is dependent on the site where the probe is placed, dynamic molecular events unselectively appear in detail in an AFM movie, facilitating our understanding of how biological molecules function. Here we present protocols for HS-AFM imaging of proteins in action, including preparation of cantilever tips, step-by-step procedures for HS-AFM imaging, and recycling of cantilevers and sample stages, together with precautions and troubleshooting advice for successful imaging. The protocols are adaptable in general for imaging many proteins and protein-nucleic acid complexes, and examples are described for looking at walking myosin, ATP-hydrolyzing rotorless F(1)-ATPase and cellulose-hydrolyzing cellulase. The entire protocol takes 10-15 h, depending mainly on the substrate surface to be used.     

Visualization of intrinsically disordered proteins by high-speed atomic force microscopy

High-speed atomic force microscopy (HS-AFM) is a powerful tool established 13 years ago. This methodology can capture individual protein molecules carrying out functional activities under near-physiological conditions, without chemical labeling, at 2–3 nm lateral and ∼0.1 nm vertical spatial resolution, and at sub-100 ms temporal resolution. Although most biological HS-AFM studies thus far target structured proteins, HS-AFM is also ideally suited to study the dynamics of intrinsically disordered proteins. Here we review some of the dynamic structures and processes of intrinsically disordered proteins that have been unveiled by HS-AFM imaging.     

Contact-Mode High-Resolution High-Speed Atomic Force Microscopy Movies of the Purple Membrane

High-speed atomic force microscopy (HS-AFM) is becoming a reference tool for the study of dynamic biological processes. The spatial and time resolutions of HS-AFM are on the order of nanometers and milliseconds, respectively, and allow structural and functional characterization of biological processes at the single-molecule level. In this work we present contact-mode HS-AFM movies of purple membranes containing two-dimensional arrays of bacteriorhodopsin (bR). In high-resolution movies acquired at a 100 ms frame acquisition time, the substructure on individual bR trimers was visualized. In regions in between different bR arrays, dynamic topographies were observed and interpreted as motion of the bR trimers. Similarly, motion of bR monomers in the vicinity of lattice defects in the purple membrane was observed. Our findings indicate that the bR arrays are in a mobile association-dissociation equilibrium. HS-AFM on membranes provides novel perspectives for analyzing the membrane diffusion processes of nonlabeled molecules.     

An overview of the biophysical applications of atomic force microscopy

The potentialities of the atomic force microscopy (AFM) make it a tool of undeniable value for the study of biologically relevant samples. AFM is progressively becoming a usual benchtop technique. In average, more than one paper is published every day on AFM biological applications. This figure overcomes materials science applications, showing that 17 years after its invention, AFM has completely crossed the limits of its traditional areas of application. Its potential to image the structure of biomolecules or bio-surfaces with molecular or even sub-molecular resolution, study samples under physiological conditions (which allows to follow in situ the real time dynamics of some biological events), measure local chemical, physical and mechanical properties of a sample and manipulate single molecules should be emphasized.     

Protein dynamics by the combination of high-speed AFM and computational modeling

High-speed atomic force microscopy (HS-AFM) allows direct observation of biological molecules in dynamic action. However, HS-AFM has no atomic resolution. This article reviews recent progress of computational methods to infer high-resolution information, including the construction of 3D atomistic structures, from experimentally acquired resolution-limited HS-AFM images.     

Dynamics of nucleosomes assessed with time-lapse high-speed atomic force microscopy

A fundamental challenge of gene regulation is the accessibility of DNA within nucleosomes. Recent studies performed by various techniques, including single-molecule approaches, led to the realization that nucleosomes are quite dynamic rather than static systems, as they were once considered. Direct data are needed to characterize the dynamics of nucleosomes. Specifically, if nucleosomes are dynamic, the following questions need to be answered. What is the range of nucleosome dynamics? Is a non-ATP-dependent unwrapping of nucleosomes possible? What are the factors facilitating the large-scale opening and unwrapping of nucleosomes? In previous studies using time-lapse atomic force microscopy (AFM) imaging, we were able, for the first time, to observe spontaneous, ATP-independent unwrapping of nucleosomes. However, low temporal resolution did not allow visualization of various pathways of nucleosome dynamics. In the studies described here, we applied high-speed time-lapse AFM (HS-AFM) capable of visualizing molecular dynamics on the millisecond time scale to study the nucleosome dynamics. The mononucleosomes were assembled on a 353 bp DNA substrate containing nucleosome-specific 601 sequence. With HS-AFM, we were able to observe the dynamics of nucleosome on a subsecond time scale and visualize various pathways of nucleosome dynamics, such as sliding and unwrapping to various extents, including complete dissociation. These studies highlight an important role of electrostatic interactions in chromatin dynamics. Overall, our findings shed new light on nucleosome dynamics and provide a novel hypothesis for the mechanisms controlling the spontaneous dynamics of chromatin.     

Tobacco mosaic virus as an AFM tip calibrator

The study of high-resolution topographic surfaces of isolated single molecules is one of the applications of atomic force microscopy (AFM). Since tip-induced distortions are significant in topographic images the exact AFM tip shape must be known in order to correct dilated AFM height images using mathematical morphology operators. In this work, we present a protocol to estimate the AFM tip apex radius using tobacco mosaic virus (TMV) particles. Among the many advantages of TMV, are its non-abrasivity, thermal stability, bio-compatibility with other isolated single molecules and stability when deposited on divalent ion pretreated mica. Compared to previous calibration systems, the advantage of using TMV resides in our detailed knowledge of the atomic structure of the entire rod-shaped particle. This property makes it possible to interpret AFM height images in term of the three-dimensional structure of TMV. Results obtained in this study show that when a low imaging force is used, the tip is sensing viral protein loops whereas at higher imaging force the tip is sensing the TMV particle core. The known size of the TMV particle allowed us to develop a tip-size estimation protocol which permits the successful erosion of tip-convoluted AFM height images. Our data shows that the TMV particle is a well-adapted calibrator for AFM tips for imaging single isolated biomolecules. The procedure developed in this study is easily applicable to any other spherical viral particles.     

Fabrication of a new substrate for atomic force microscopic observation of DNA molecules from an ultrasmooth sapphire plate.

A new stable substrate applicable to the observation of DNA molecules by atomic force microscopy (AFM) was fabricated from a ultrasmooth sapphire (alpha-Al2O3 single crystal) plate. The atomically ultrasmooth sapphire as obtained by high-temperature annealing has hydrophobic surfaces and could not be used for the AFM observation of DNA. However, sapphire treated with Na3PO4 aqueous solution exhibited a hydrophilic character while maintaining a smooth surface structure. The surface of the wet-treated sapphire was found by x-ray photoelectron spectroscopy and AFM to be approximately 0.3 nm. The hydrophilic surface character of the ultrasmooth sapphire plate made it easy for DNA molecules to adhere to the plate. Circular molecules of the plasmid DNA could be imaged by AFM on the hydrophilic ultrasmooth sapphire plate.     

Direct visualization of avian influenza H5N1 hemagglutinin precursor and its conformational change by high-speed atomic force microscopy

BackgroundHemagglutinin (HA) of influenza A is one of the key virulence factors that mediates the release of viral components in host cells. HA is initially synthesized as a trimeric precursor (HA0) and then it is cleaved by proteases to become a functional HA. Low pH induces irreversible conformational changes in both HA0 and HA but only HA is fusion compatible. Here, we used high-speed atomic force microscopy (HS-AFM) to record conformational changes in HA0 trimers (H5N1) from neutral to acidic conditions at a millisecond scale.MethodsPurified HA0 protein was diluted with either neutral Tris-HCl (pH 7.4) or acetic acid-titrated Tris-HCl (pH 5.0) and then loaded onto bare mica. Neutral or acidic Tris-HCl was used as the scanning buffer. HS-AFM movies were recorded and processed using Image J software.ResultsThe conformation of HA0_neutral visualized using HS-AFM was comparable to the HA trimer structures depicted in the PDB data and the AFM simulator. HA0 underwent rapid conformational changes under low pH condition. The circularity and area of HA0_acid were significantly higher than in HA0_neutral. In contrast, the height of HA0_acid was significantly lower than in HA0_neutral.ConclusionsWe have captured real-time images of the native HA0 trimer structure under physiological conditions using HS-AFM. By analyzing the images, we confirm that HA0 trimer is sensitive to acidic conditions.General significanceThe dynamic nature of the HA structure, particularly in the host endosome, is essential for H5N1 infectivity. Understanding this acidic behavior is imperative for designing therapeutic strategies against H5N1. This article reports a sophisticated new tool for studying the spatiotemporal dynamics of the HA precursor protein.     

Real-Time Dynamic Adsorption Processes of Cytochrome c on an Electrode Observed through Electrochemical High-Speed Atomic Force Microscopy

An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome c (cyt c) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt c adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt c molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt c using EC-HS-AFM. The root mean square roughness (R_RMS) from the AFM images and the number of the electrochemically active cyt c molecules adsorbed onto the electrode (Γ) simultaneously increased in positive cooperativity. Cyt c molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.     

Observing single biomolecules at work with the atomic force microscope

Progress in the application of the atomic force microscope (AFM) to imaging and manipulating biomolecules is the result of improved instrumentation, sample preparation methods and image acquisition conditions. Biological membranes can be imaged in their native state at a lateral resolution of 0.5-1 nm and a vertical resolution of 0. 1-0.2 nm. Conformational changes that are related to functions can be resolved to a similar resolution, complementing atomic structure data acquired by other methods. The unique capability of the AFM to directly observe single proteins in their native environments provides insights into the interactions of proteins that form functional assemblies. In addition, single molecule force spectroscopy combined with single molecule imaging provides unprecedented possibilities for analyzing intramolecular and intermolecular forces. This review discusses recent examples that illustrate the power of AFM.     

Molecular basis of fibrin clot elasticity

Blood clots must be stiff to stop hemorrhage yet elastic to buffer blood's shear forces. Upsetting this balance results in clot rupture and life-threatening thromboembolism. Fibrin, the main component of a blood clot, is formed from molecules of fibrinogen activated by thrombin. Although it is well known that fibrin possesses considerable elasticity, the molecular basis of this elasticity is unknown. Here, we use atomic force microscopy (AFM) and steered molecular dynamics (SMD) to probe the mechanical properties of single fibrinogen molecules and fibrin protofibrils, showing that the mechanical unfolding of their coiled-coil alpha helices is characterized by a distinctive intermediate force plateau in the systems' force-extension curve. We relate this plateau force to a stepwise unfolding of fibrinogen's coiled alpha helices and of its central domain. AFM data show that varying pH and calcium ion concentrations alters the mechanical resilience of fibrinogen. This study provides direct evidence for the coiled alpha helices of fibrinogen to bring about fibrin elasticity.     

Quantum dot binding to DNA: Single-molecule imaging with atomic force microscopy

The interaction between nanoparticles (NPs) and DNA is of significance for both application and implication research of NPs. In this study, a single-molecule imaging technique based on atomic force microscopy (AFM) was employed to probe the NP-DNA interactions with quantum dots (QDs) as model NPs. Reproducible high-quality images of single DNA molecules in air and in liquids were acquired on mica by optimizing sample preparation conditions. Furthermore, the binding of QDs to DNA was explored using AFM. The DNA concentration was found to be a key factor influencing AFM imaging quality. In air and liquids, the optimal DNA concentration for imaging DNA molecules was approximately 2.5 and 0.25 μg/mL, and that for imaging DNA binding with QDs was 0.5 and 0.25 μg/mL, respectively. In the presence of QDs, the DNA conformation was altered with the formation of DNA condensates. Finally, the fine conformation of QD-DNA binding sites was examined to analyze the binding mechanisms. This work will benefit investigations of NP-DNA interactions and the understanding of the structure of NP-DNA bioconjugates. See accompanying article by Wang DOI: 10.1002/biot.201200309     

原子力显微技术在酶学研究中的应用

酶在生物体的生命活动中占有及其重要的地位,机体功能的和谐统一有赖于酶的作用。原子力显微技术(AFM)作为一门新发展起来的技术,为人们认识酶的结构与功能提供了又一新的窗口。AFM能够在生理条件下对生物样品进行三维成像,在分子水平上实时监测生理生化反应。AFM还能够在皮牛顿精度上测定分子间作用力。目前,AFM已用于单分子酶的化学性质及其作用原理的研究。本简述AFM在酶学中的应用情况。