基于原子力显微镜的单分子力谱在生物研究中的应用

原子力显微镜被广泛应用于生物研究领域,基于原子力显微镜的单分子力谱可以在单分子、单细胞水平上研究生物分子内和分子间的相互作用。 本文介绍了原子力显微镜单分子力谱在生物分子间相互作用、蛋白质去折叠、细胞表面生物分子、细胞力学性质和基于单分子力谱成像等研究中的最新进展。     

Quantitative mapping of elastic moduli at the nanoscale in phase separated polyurethanes by AFM

The micro phase separated nanoscale morphology of phase separated polyurethanes (PUs) was visualized by atomic force microscopy (AFM) height and phase imaging of smooth surfaces obtained by ultramicrotonomy. PUs were obtained from 4,4′-methylenbis (phenyl isocyanate) (MDI), 1,4-butanediol (BD) and poly(tetrahydrofurane) polyether polyol (PTHF). The segmented polyether PUs with varying stoichiometric ratio of the isocyanate and hydroxyl groups were prepared to investigate the effect of molar mass, as well as the type and number of end-groups on their morphology and mechanical performance.The PU samples studied show characteristic “fingerprint” AFM phase images. Novel dynamic imaging modes of AFM, including HarmoniX material mapping and Peak Force Tapping were used to assess the mechanical performance of phase separated polyurethanes quantitatively as a function of their molecular structure. The values of surface elastic moduli were determined with nanoscale resolution and were in excellent agreement for both AFM modes. While tensile testing provides a bulk average value for the elastic modulus of the elastomers, the novel AFM based elastic moduli mappings introduced enable the study of surface stiffness with nanoscale resolution in a quantitative way.     

Bulk phase separation study by atomic force microscope friction imaging of natural rubber/poly(methyl methacrylate) film

The first observation of bulk phase separation in immiscible natural rubber (NR)/poly(methyl methacrylate) (PMMA) film using atomic force microscopy (AFM) is reported. Three different forms of AFM measurements: topographic, friction force imaging, and nanoindentation have been effectively used to investigate combined morphological and compositional mapping of the NR/PMMA system. The fracture temperature during sample microtoming and material physical properties could be responsible for the observed topographic contrast. The stronger contrast of friction imaging, relative to topographic imaging, is ascribed to local variations in mechanical properties of the phase-separated domains. Friction force imaging associated with nanoindentation response, performed under AFM force mode, highlights the AFM's ability for probing local friction, adhesion, and elastic properties, and for compositional mapping of heterogeneous polymer film. The resulting friction force imaging along with the response of the nanoindentation are in good agreement, indicating that PMMA exists mainly near the modified NR surface.     

Investigating the morphology and mechanical properties of blastomeres with atomic force microscopy

Atomic force microscopy (AFM) was used to directly investigate the morphology and mechanical properties of blastomeres during the embryo development. With AFM imaging, the surface topography of blastomeres from two‐cell, four‐cell, and eight‐cell stages was visualized, and the AFM images clearly revealed the blastomere's morphological changes during the different embryo developmental stages. The section measurements of the AFM topography images of the blastomeres showed that the axis of the embryos nearly kept constant during the two‐cell, four‐cell, and eight‐cell stages. With AFM indenting, the mechanical properties of living blastomeres from several embryos were measured quantitatively under physiological conditions. The results of mechanical properties measurements indicated that the Young's modulus of the two blastomeres from two‐cell embryo was different from each other, and the four blastomeres from the four‐cell embryo also had variable Young's modulus. Besides, the blastomeres from two‐cell embryos were significantly harder than blastomeres from four‐cell embryos. These results can improve our understanding of the embryo development from the view of cell mechanics. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of the physical properties of model biomembranes at the nanometer scale with the atomic force microscope

Interaction forces and topography of mixed phospholipid-glycolipid bilayers were investigated by atomic force microscopy (AFM) in aqueous conditions with probes functionalized with self-assembled monolayers terminating in hydroxy groups. Short-range repulsive forces were measured between the hydroxy-terminated probe and the surface of the two-dimensional (2-D) solid-like domains of distearoyl-phosphatidylethanolamine (DSPE) and digalactosyldiglyceride (DGDG). The form and range of the short-range repulsive force indicated that repulsive hydration/steric forces dominate the interaction at separation distances of 0.3-1.0 nm after which the probe makes mechanical contact with the bilayers. At loads < 5 nN the bilayer was elastically deformed by the probe, while at higher loads plastic deformation of the bilayer was observed. Surprisingly, a short-range repulsive force was not observed at the surface of the 2-D liquid-like dioleoylphosphatidylethanolamine (DOPE) film, despite the identical head groups of DOPE and DSPE. This provides direct evidence for the influence of the structure and mechanical properties of lipid bilayers on their interaction forces, an effect which may be a major importance in the control of biological processes such as cell adhesion and membrane fusion. The step height measured between lipid domains in the AFM topographic images was larger than could be accounted for by the thickness and mechanical properties of the molecules. A direct correlation was observed between the repulsive force range over the lipid domains and the topographic contrast, which provides direct insight into the fundamental mechanisms of AFM imaging in aqueous solutions. This study demonstrates that chemically modified AFM probes can be used in combination with patterned lipid bilayers as a novel and powerful approach to characterize the nanometer scale chemical and physical properties of heterogeneous biosurfaces such as cell membranes.     

原子力显微镜在生物大分子结构研究中的应用进展

评述了原子力显微镜（ａｔｏｍｉｃ ｆｏｒｃｅ ｍｉｃｒｏｓｃｏｐｙ,ＡＦＭ）的成镜模式与探针技术的发展以及在脱氧核糖核酸、蛋白质和多糖等生物大分子结构研究中的应用进展,并展望了原子力显微镜在此领域的发展前景。     

Combined AFM and two-focus SFCS study of raft-exhibiting model membranes.

Dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/SM/cholesterol) model membranes exhibit liquid-liquid phase separation and therefore provide a physical model for the putative liquid-ordered domains present in cells. Here we present a combination of atomic force microscopy (AFM) imaging, force measurements, confocal fluorescence imaging and two-focus scanning fluorescence correlation spectroscopy (two-focus SFCS) to obtain structural and dynamical information about this model membrane system. Partition coefficients and diffusion coefficients in the different phases were measured with two-focus SFCS for numerous fluorescent lipid analogues and proteins, while being directly related to the lateral organization of the membrane and its mechanical properties probed by AFM. Moreover we show how the combination of these different approaches is effective in reducing artifacts resulting from the use of a single technique.     

Three-dimensional morphological characterization of optic nerve fibers by atomic force microscopy and by scanning electron microscopy.

A comparative study of scanning electron microscopy (SEM) and atomic force microscopy (AFM) imaging of the healthy human optic nerve was carried out to determine the similarities and the differences. In this study we compared the fine optic nerve structures as observed by SEM and AFM. The fibers of the right optic nerve of a 61-year-old man show different arrangements in transverse sections taken from the same individual 5 mm central to the optic canal and 5 mm peripheral to the optic chiasma; this difference can be recognized by light microscopy (LM), SEM, and AFM. AFM revealed such typical optic nerve fibers (taken from a point 5 mm central to the optic canal) with annular and longitudinal orientations, which were not visible by SEM in this form. By contrast, LM and SEM visualized other structures, such as pia mater and optic nerve fibers loosely arranged in bundles, none of which was visualized by AFM. The images, however, taken 5 mm peripheral from the optic chiasma show shapeless nerve fibers having a wavy course. Our results reveal that more detailed information on optic nerve morphology is obtained by exploiting the advantages of both SEM and AFM. These are the first SEM and AFM images of healthy human optic nerve fibers, containing clear representations of the three dimensions of the optic nerve.     

Characterization of fiber-forming peptides and proteins by means of atomic force microscopy

The atomic force microscope (AFM) is widely used in biological sciences due to its ability to perform imaging experiments at high resolution in a physiological environment, without special sample preparation such as fixation or staining. AFM is unique, in that it allows single molecule information of mechanical properties and molecular recognition to be gathered. This review sets out to identify methodological applications of AFM for characterization of fiber-forming proteins and peptides. The basics of AFM operation are detailed, with in-depth information for any life scientist to get a grasp on AFM capabilities. It also briefly describes antibody recognition imaging and mapping of nanomechanical properties on biological samples. Subsequently, examples of AFM application to fiber-forming natural proteins, and fiber-forming synthetic peptides are given. Here, AFM is used primarily for structural characterization of fibers in combination with other techniques, such as circular dichroism and fluorescence spectroscopy. More recent developments in antibody recognition imaging to identify constituents of protein fibers formed in human disease are explored. This review, as a whole, seeks to encourage the life scientists dealing with protein aggregation phenomena to consider AFM as a part of their research toolkit, by highlighting the manifold capabilities of this technique.     

原子力显微镜与透射电镜对比研究嵌段共聚物微相分离形态

嵌段共聚物微观相分离形态结构由于其理论与应用中的重要性一直是人们研究的热点 [1] .本文研究的聚苯乙烯 -聚乙烯 /聚丁烯 -聚苯乙烯三嵌段共聚物 (SEBS)为聚苯乙烯 -聚丁二烯 -聚苯乙烯 (SBS)饱和加氢后的产物 .由于中间嵌段中的双键大多已加氢饱和 ,过去一直没有合适的染色剂进行染色 ,所以在 2 0世纪 80年代中期以前 ,SEBS形态结构未见文献报道 [1] .近年来 ,由于新型染色剂 Ru O4 的应用 ,使得用透射电镜 (TEM)观察其微相分离形态成为可能 .　　随着对嵌段共聚物形态结构认识程度深入 ,人们迫切希望能有新的手段提供更加丰富的…     

Study of amyloid fibrils via atomic force microscopy

Protein fibrils are a crucial subject of study in various research fields and disciplines. Amyloid fibrils are highly ordered fibrillar structures assembled from either peptides or unfolded proteins, which have a great importance in biology, medicine and recently have started to find an important role in many nanotechnology applications. Understanding the mechanisms of fibrillation, the structural features, and the physical and mechanical properties of these fibrils is an essential step to both unraveling their biological role and also their successful applications in nanotechnology and material science. Atomic force microscopy (AFM) is one of the most widely used single-molecule techniques to study the properties of amyloid fibrils. In this review we will discuss how the application of AFM during last few years has allowed moving considerably forward in the research of amyloid fibrils. We will review how AFM has rapidly evolved from a purely microscopic technique, providing important information about fibril structure and fibrillation processes, to a tool capable to probe also intrinsic properties of amyloid fibrils such as their strength and Young's moduli.     

Comparison of scanning ion conductance microscopy with atomic force microscopy for cell imaging

We present the first direct comparison of scanning ion conductance microscopy (SICM) with atomic force microscopy (AFM) for cell imaging. By imaging the same fibroblast or myoblast cell with both technologies in series, we highlight their advantages and disadvantages with respect to cell imaging. The finite imaging force applied to the sample in AFM imaging results in a coupling of mechanical sample properties into the measured sample topography. For soft samples such as cells this leads to artifacts in the measured topography and to elastic deformation, which we demonstrate by imaging whole fixed cells and cell extensions at high resolution. SICM imaging, on the other hand, has a noncontact character and can provide the true topography of soft samples at a comparable resolution.     

A Chemo‐Mechanical Tweezer for Single‐Molecular Characterization of Soft Materials

A new atomic force microscopy (AFM)‐based chemo‐mechanical tweezer has been developed that can measure mechanical properties of individual macromolecules in supramolecular assembly and reveal positions of azide‐containing polymers. A key feature of the new technology is the use of an AFM tip densely modified with 4‐dibenzocyclooctynols (chemo‐mechanical tweezer) that can react with multiple azide containing macromolecules of micelles to give triazole “clicked” compounds, which during retracting phases of AFM imaging are removed from the macromolecular assembly thereby providing a surface topographical image and positions of azide‐containing polymers. The force–distance curves gave mechanical properties of removal of individual molecules from a supramolecular assembly. The new chemo‐mechanical tweezer will make it possible to characterize molecular details of macromolecular assemblies thereby offering new avenues to tailor properties of such assemblies.     

A novel single-molecule study to determine protein--protein association constants

Atomic force microscopy (AFM) is traditionally used as an imaging technique to gain qualitative information for a biological system. We have successfully used the imaging capabilities of the AFM to determine protein-protein association constants. We have developed a method to measure the molecular weight of a protein based on its volume determined from AFM images. Our volume determination method allows for rapid, accurate analysis of large protein populations. On the basis of the measured volume, the fraction of monomers as dimers was determined for the DNA helicase UvrD, and the dissociation constant (K(d)) for the helicase was calculated. We determined a K(d) for UvrD of 1.4 microM, which is in good agreement with published K(d) data obtained from analytical ultracentrifugation (AUC) studies. Our method provides a rapid method for determining protein-protein association constants.     

Comparative study of nanoscale surface structures of calcite microcrystals using FE-SEM, AFM, and TEM.

The bulk morphology and surface features that developed upon precipitation on micrometer-size calcite powders and millimeter-size cleavage fragments were imaged by three different microscopic techniques: field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM) of Pt-C replicas, and atomic force microscopy (AFM). Each technique can resolve some nanoscale surface features, but they offer different ranges of magnification and dimensional resolutions. Because sample preparation and imaging is not constrained by crystal orientation, FE-SEM and TEM of Pt-C replicas are best suited to image the overall morphology of microcrystals. However, owing to the decoration effect of Pt-C on the crystal faces, TEM of Pt-C replicas is superior at resolving nanoscale surface structures, including the development of new faces and the different microtopography among nonequivalent faces in microcrystals, which cannot be revealed by FE-SEM. In conjunction with SEM, Pt-C replica provides the evidence that crystals grow in diverse and face-specific modes. The TEM imaging of Pt-C replicas has nanoscale resolution comparable to AFM. AFM yielded quantitative information (e.g., crystallographic orientation and height of steps) of microtopographic features. In contrast to Pt-C replicas and SEM providing three-dimensional images of the crystals, AFM can only image one individual cleavage or flat surface at a time.     

A chemo-mechanical tweezer for single-molecular characterization of soft materials

A new atomic force microscopy (AFM)-based chemo-mechanical tweezer has been developed that can measure mechanical properties of individual macromolecules in supramolecular assembly and reveal positions of azide-containing polymers. A key feature of the new technology is the use of an AFM tip densely modified with 4-dibenzocyclooctynols (chemo-mechanical tweezer) that can react with multiple azide containing macromolecules of micelles to give triazole "clicked" compounds, which during retracting phases of AFM imaging are removed from the macromolecular assembly thereby providing a surface topographical image and positions of azide-containing polymers. The force-distance curves gave mechanical properties of removal of individual molecules from a supramolecular assembly. The new chemo-mechanical tweezer will make it possible to characterize molecular details of macromolecular assemblies thereby offering new avenues to tailor properties of such assemblies.     

Correlated atomic force microscopy and fluorescence lifetime imaging of live bacterial cells

We report on imaging living bacterial cells by using a correlated tapping-mode atomic force microscopy (AFM) and confocal fluorescence lifetime imaging microscopy (FLIM). For optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells, we explored different methods of bacterial sample preparation, such as spreading the cells on poly-L-lysine coated surfaces or agarose gel coated surfaces. We have found that the agarose gel containing 99% ammonium acetate buffer can provide sufficient local aqueous environment for single bacterial cells. Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded. Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and signal-to-noise ratio of the AFM images. Near-field AFM-tip-enhanced fluorescence lifetime imaging (AFM-FLIM) holds high promise on obtaining fluorescence images beyond optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond diffraction limited spatial resolution. Here, as the first step of applying AFM-FLIM on imaging bacterial living cells, we demonstrated a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging of living bacterial cells to characterize cell polarity.     

Enhanced mechanical stability of microtubules polymerized with a slowly hydrolyzable nucleotide analogue

Atomic force microscopy (AFM) has been used to investigate the local mechanical and structural properties of microtubules polymerized using guanylyl-alpha-beta-methylene diphosphonate (GMPCPP), a slowly hydrolyzable analogue of guanosine triphosphate. Using a combination of AFM imaging and local force spectroscopy, GMPCPP-polymerized microtubules have been qualitatively and quantitatively compared to paclitaxel-stabilized microtubules. GMPCPP-polymerized microtubules qualitatively display a greater resistance to destruction by the AFM probe tip during imaging and during deformation measurements and maintain structural details after indentation. In addition, using force spectroscopy taken during the indentation and collapse of individual microtubules with the AFM probe tip, an effective spring constant of the microtubule wall (kMT) for both types of microtubules was determined. The average kMT of GMPCPP-polymerized microtubules, 0.172 N/m, is more than twice that of paclitaxel-stabilized microtubules. These results complement previously reported measurements of bending experiments on GMPCPP-polymerized and paclitaxel-stabilized microtubules.     

利用AFM和SNOM对淋巴细胞膜表面超微结构及其光学性质的初步研究

利用原子力显微镜(Atomic Force Microscopy，AFM)对淋巴细胞表面形貌进行了形态学的初步研究，观察到了其膜表面其他显微技术所不能发现的超微结构．同时也运用扫描近场光学显微镜(Scanning Near field Optical Microscopy，SNOM)对淋巴细胞进行成像，观察了其对光的透射、吸收等光学性质，并对两种成像方法进行了比较．研究发现：淋巴细胞膜表面凹凸不平，分布着大量直径几十到几百纳米不等的小颗粒；淋巴细胞中央部位有自发荧光现象．结果表明，AFM和SNOM可作为进一步探讨淋巴细胞的结构与功能关系的有力工具．