Imaging biological structures with the cryo atomic force microscope.

It has long been recognized that one of the major limitations in biological atomic force microscopy (AFM) is the softness of most biological samples, which are easily deformed or damaged by the AFM tip, because of the high pressure in the contact area, especially from the very sharp tips required for high resolution. Another is the molecular motion present at room temperature due to thermal fluctuation. Using an AFM operated in liquid nitrogen vapor (cryo-AFM), we demonstrate that cryo-AFM can be applied to a large variety of biological samples, from immunoglobulins to DNA to cell surfaces. The resolution achieved with cryo-AFM is much improved when compared with AFM at room temperature with similar specimens, and is comparable to that of cryo-electron microscopy on randomly oriented macromolecules. We will also discuss the technical problems that remain to be solved for achieving even higher resolution with cryo-AFM and other possible applications of this novel technique.     

New technologies in scanning probe microscopy for studying molecular interactions in cells

Atomic force microscopy (AFM) is a specialised form of scanning probe microscopy, which was invented by Binnig and colleagues in 1986. Since then, AFM has been increasingly used to study biomedical problems. Because of its high resolution, AFM has been used to examine the topography or shape of surfaces, such as during the molecular imaging of proteins. This, combined with the ability to operate under known force regimes, makes AFM technology particularly useful for measuring intermolecular bond forces and assessing the mechanical properties of biological materials. Many of the constraints (e.g. complex instrumentation, slow acquisition speeds and poor vertical range) that previously limited the use of AFM in cell biology are now beginning to be resolved. Technological advances will enable AFM to challenge both confocal laser scanning microscopy and scanning electron microscopy as a method for carrying out three-dimensional imaging. Its use as both a precise micro-manipulator and a measurement tool will probably result in many novel and exciting applications in the future. In this article, we have reviewed some of the current biological applications of AFM, and illustrated these applications using studies of the cell biology of bone and integrin-mediated adhesion.     

原子力显微镜对纳米生物结构的观察和操纵

原子力显微镜不仅能对纳米生物结构进行观察,而且也能对其进行操纵。对纳米生物结构的观察已深入到生物大分子结构水平。原子力显微镜对生物大分子的操纵包括从染色质中提取DNA用于基因分析、对膜蛋白的结构进行观察、对蛋白构象进行可控操纵等。这些纳米技术的应用将揭示生物系统更多的结构和功能信息。     

Atomic force microscopy: from cellular imaging to molecular manipulation

Using a sharp tip attached at the end of a soft cantilever as a probe, the atomic force microscope (AFM) explores the surface topography of biological samples bathed in physiological solutions. In the last few years, the AFM has gained popularity among biologists. This has been obtained through the improvement of the equipment and imaging techniques as well as through the development of new non-imaging applications. Biological imaging has to face a main difficulty that is the softness and the dynamics of most biological materials. Progress in understanding the AFM tip-biological samples interactions provided spectacular results in different biological fields. Recent examples of the possibilities offered by the AFM in the imaging of intact cells, isolated membranes, membrane model systems and single molecules at work are discussed in this review. Applications where the AFM tip is used as a nanotool to manipulate biomolecules and to determine intra- and intermolecular forces from single molecules are also presented.     

Biomolecular imaging using atomic force microscopy

Atomic force microscopy (AFM) has become a well-established technique for imaging single biomacromolecules under physiological conditions. The exceptionally high spatial resolution and signal-to-noise ratio of the AFM enables the substructure of individual molecules to be observed. In contrast to other methods, specimens prepared for AFM remain in a plastic state, which enables direct observation of the dynamic molecular response, creating unique opportunities for studying the structure–function relationships of proteins and their functionally relevant assemblies. This review presents recent advances in methods and applications of AFM to imaging biological samples. It is clear that AFM will become an increasingly important tool for probing both the structural and kinetic properties of biological macromolecules.     

Atomic Force Microscopy and pharmacology: From microbiology to cancerology

Background

Atomic Force Microscopy (AFM) has been extensively used to study biological samples. Researchers take advantage of its ability to image living samples to increase our fundamental knowledge (biophysical properties/biochemical behavior) on living cell surface properties, at the nano-scale.

Scope of review

AFM, in the imaging modes, can probe cells morphological modifications induced by drugs. In the force spectroscopy mode, it is possible to follow the nanomechanical properties of a cell and to probe the mechanical modifications induced by drugs. AFM can be used to map single molecule distribution at the cell surface. We will focus on a collection of results aiming at evaluating the nano-scale effects of drugs, by AFM. Studies on yeast, bacteria and mammal cells will illustrate our discussion. Especially, we will show how AFM can help in getting a better understanding of drug mechanism of action.

Major conclusions

This review demonstrates that AFM is a versatile tool, useful in pharmacology. In microbiology, it has been used to study the drugs fighting Candida albicans or Pseudomonas aeruginosa. The major conclusions are a better understanding of the microbes' cell wall and of the drugs mechanism of action. In cancerology, AFM has been used to explore the effects of cytotoxic drugs or as an innovative diagnostic technology. AFM has provided original results on cultured cells, cells extracted from patient and directly on patient biopsies.

General significance

This review enhances the interest of AFM technologies for pharmacology. The applications reviewed range from microbiology to cancerology.     

Surface topography of membrane domains

Elucidating origin, composition, size, and lifetime of microdomains in biological membranes remains a major issue for the understanding of cell biology. For lipid domains, the lack of a direct access to the behaviour of samples at the mesoscopic scale has constituted for long a major obstacle to their characterization, even in simple model systems made of immiscible binary mixtures. By its capacity to image soft surfaces with a resolution that extends from the molecular to the microscopic level, in air as well as under liquid, atomic force microscopy (AFM) has filled this gap and has become an inescapable tool in the study of the surface topography of model membrane domains, the first essential step for the understanding of biomembranes organization. In this review we mainly focus on the type of information on lipid microdomains in model systems that only AFM can provide. We will also examine how AFM can contribute to understand data acquired by a variety of other techniques and present recent developments which might open new avenues in model and biomembrane AFM applications.     

Heterogeneous cell mechanical properties: an atomic force microscopy study.

Atomic force microscopy (AFM) is a non-invasive microscopy to explore living biological systems like cells in liquid environment. Thus AFM is an appropriate tool to investigate surface chemical modification and its influence on biological systems. In particular, control over biomaterial surface chemistry can result in a regulated cell response. This report investigates the influence of adhesive and non-adhesive surfaces on the cell morphology and the influence of the cytoskeleton structure on the local mechanical properties. In this study, the main work concerns a thorough investigation of the height images obtained with an AFM as therecorded images provide the evolution of the mechanical properties of the cell as function of its local structure. Information on the cell elasticity due to the cytoskeleton organization is deduced when comparing the AFM tip indentation depth versus the distance between the cytoskeleton bundles for the different samples.     

High-speed atomic force microscopy for observing dynamic biomolecular processes

The atomic force microscope (AFM) is unique in its capability to capture high-resolution images of biological samples in liquids. This capability will become more valuable to biological sciences if AFM additionally acquires an ability of high-speed imaging, because 'direct and real-time visualization' is a straightforward and powerful means to understand biomolecular processes. With conventional AFMs, it takes more than a minute to capture an image, while biomolecular processes generally occur on a millisecond timescale or less. In order to fill this large gap, various efforts have been carried out in the past decade. Here, we review these past efforts, describe the current state of the capability and limitations of high-speed AFM, and discuss possibilities that may break the limitations and lead to the development of a truly useful high-speed AFM for biological sciences.     

原子力显微镜在生物学研究中的应用进展

原子力显微镜（atomic force microscope,AFM)具有原子级分辨率,能够在生理条件下对生物样品进行观察,本综述了AFM的原理及技术要点,举例说明了它在核酸,蛋白质,微生物及细胞等领域的应用进展,相信AFM必将在生物学研究中起到越来越重要的作用。     

Application of atomic force microscopy to the study of micromechanical properties of biological materials

Atomic force microscopy (AFM) has been used to study the micromechanical properties of biological systems. Its unique ability to function both as an imaging device and force sensor with nanometer resolution in both gaseous and liquid environments has meant that AFM has provided unique insights into the mechanical behaviour of tissues, cells and single molecules. As a surface scanning device, AFM can map properties such as adhesion and the Young's modulus of surfaces. As a force sensor and nanoindentor AFM can directly measure properties such as the Young's modulus of surfaces or the binding forces of cells. As a stress-strain gauge AFM can study the stretching of single molecules or fibres and as a nanomanipulator it can dissect biological particles such as viruses or DNA strands. The present paper reviews key research that has demonstrated the versatility of AFM and how it can be exploited to study the micromechanical behaviour of biological materials.     

The application of atomic force microscopy to the study of living vertebrate cells in culture

Atomic force microscopy (AFM), a relatively new variant of scanning probe microscopy developed for the material sciences, is becoming an increasingly important tool in other disciplines. In this review I describe in nontechnical terms some of the basic aspects of using AFM to study living vertebrate cells. Although AFM has some unusual attributes such as an ability to be used with living cells, AFM also has attributes that make its use in cell biology a real challenge. This review was written to encourage researchers in the biological and biomedical sciences to consider AFM as a potential (and potent) tool for their cell biological research.     

FM-AFM constant height imaging and force curves: high resolution study of DNA-tip interactions

Interaction of the atomic force microscopy (AFM) tip with the sample can be invasive for soft samples. Frequency Modulation (FM) AFM is gentler because it allows scanning in the non‐contact regime where only attractive forces exist between the tip and the sample, and there is no sample compression. Recently, FM‐AFM was used to resolve the atomic structure of single molecules of pentacene and of carbon nanotubes. We are testing similar FM‐AFM‐based approaches to study biological samples. We present FM‐AFM experiments on dsDNA deposited on 3‐aminopropyltriethoxysilane modified mica in ultra high vacuum. With flexible samples such as DNA, the substrate flatness is a sub‐molecular resolution limiting factor. Non‐contact topographic images of DNA show variations that have the periodicity of the right handed helix of B‐form DNA – this is an unexpected result as dehydrated DNA is thought to assume the A‐form structure. Frequency shift maps at constant height allow working in the non‐monotonic frequency shift range, show a rich contrast that changes significantly with the tip‐sample separation, and show 0.2 to 0.4 nm size details on DNA. Frequency shift versus distance curves acquired on DNA molecules and converted in force curves show that for small molecules (height < 2.5 nm), there is a contribution to the interaction force from the substrate when the tip is on top of the molecules. Our data shine a new light on dehydrated and adsorbed DNA behavior. They show a longer tip‐sample interaction distance. These experiments may have an impact on nanotechnological DNA applications in non‐physiological environments such as DNA based nanoelectronics and nanotemplating. Copyright © 2012 John Wiley & Sons, Ltd.     

Amyloid under the atomic force microscope

The atomic force microscope (AFM) is a versatile instrument that can be used to image biological samples at nanometre resolution as well as to measure inter and intra-molecular forces in air and liquid environments. This review summarises the use of AFM applied to protein and peptide self-assembly systems involved in amyloid formation. The technical principles of the AFM are outlined and its advantages and disadvantages are highlighted and discussed in the context of the rapidly developing field of amyloid research.     

Dehomogenized Elastic Properties of Heterogeneous Layered Materials in AFM Indentation Experiments

Atomic force microscopy (AFM) is used to study mechanical properties of biological materials at submicron length scales. However, such samples are often structurally heterogeneous even at the local level, with different regions having distinct mechanical properties. Physical or chemical disruption can isolate individual structural elements but may alter the properties being measured. Therefore, to determine the micromechanical properties of intact heterogeneous multilayered samples indented by AFM, we propose the Hybrid Eshelby Decomposition (HED) analysis, which combines a modified homogenization theory and finite element modeling to extract layer-specific elastic moduli of composite structures from single indentations, utilizing knowledge of the component distribution to achieve solution uniqueness. Using finite element model-simulated indentation of layered samples with micron-scale thickness dimensions, biologically relevant elastic properties for incompressible soft tissues, and layer-specific heterogeneity of an order of magnitude or less, HED analysis recovered the prescribed modulus values typically within 10% error. Experimental validation using bilayer spin-coated polydimethylsiloxane samples also yielded self-consistent layer-specific modulus values whether arranged as stiff layer on soft substrate or soft layer on stiff substrate. We further examined a biophysical application by characterizing layer-specific microelastic properties of full-thickness mouse aortic wall tissue, demonstrating that the HED-extracted modulus of the tunica media was more than fivefold stiffer than the intima and not significantly different from direct indentation of exposed media tissue. Our results show that the elastic properties of surface and subsurface layers of microscale synthetic and biological samples can be simultaneously extracted from the composite material response to AFM indentation. HED analysis offers a robust approach to studying regional micromechanics of heterogeneous multilayered samples without destructively separating individual components before testing.     

原子力显微镜在双微体形态学研究中的应用

原子力显微术(atomic force microscopy,AFM)是一种新型的纳米显微技术,由于其拥有标本制备简单、分辨率高等优点,因此常用于细胞超微结构的观察。双微体(double minute chromosomes,DMs)是基因扩增的主要表现形式,经常出现在肿瘤细胞及耐药细胞中,可使肿瘤细胞获得生存优势或产生耐药性,因此对双微体进行研究可使人类了解肿瘤的生长特性及其抗药性的产生机理。为寻找一种研究双微体的有效方法,本实验利用原子力显微镜对小鼠耐氨甲喋呤细胞3T3R500中的双微体进行观察,在获得双微体高分辨AFM形态图的同时,还对双微体的大小进行了测量,发现细胞中双微体大小存在差异。此外,就原子力显微镜在双微体研究中的一些技术细节进行了探讨。实验结果表明原子力显微术是研究双微体的一种有效手段。     

AFM characterization of solid-supported lipid multilayers prepared by spin-coating

Lipids are the principal components of biologically relevant structures as cellular membranes. They have been the subject of many studies due to their biological relevance and their potential applications. Different techniques, such as Langmuir-Blodgett and vesicle-fusion deposition, are available to deposit ordered lipid films on etched surfaces. Recently, a new technique of lipid film deposition has been proposed in which stacks of a small and well-controlled number of bilayers are prepared on a suitable substrate using a spin-coater. We studied the morphological properties of multi-layers made of cationic and neutral lipids (DOTAP and DOPC) and mixtures of them using dynamic mode atomic force microscopy (AFM). After adapting and optimizing, the spin-coating technique to deposit lipids on a chemically etched Silicon (1,0,0) substrate, a morphological nanometer-scale characterization of the aforementioned samples has been provided. The AFM study showed that an initial layer of ordered vesicles is formed and, afterward, depending on details of the spin-coating preparation protocol and to the dimension of the silicon substrate, vesicle fusion and structural rearrangements of the lipid layers may occur. The present data disclose the possibility to control the lipid's structures by acting on spin-coating parameters with promising perspectives for novel applications of lipid films.     

AFM characterization of solid-supported lipid multilayers prepared by spin-coating

Lipids are the principal components of biologically relevant structures as cellular membranes. They have been the subject of many studies due to their biological relevance and their potential applications. Different techniques, such as Langmuir-Blodgett and vesicle-fusion deposition, are available to deposit ordered lipid films on etched surfaces. Recently, a new technique of lipid film deposition has been proposed in which stacks of a small and well-controlled number of bilayers are prepared on a suitable substrate using a spin-coater.We studied the morphological properties of multi-layers made of cationic and neutral lipids (DOTAP and DOPC) and mixtures of them using dynamic mode atomic force microscopy (AFM). After adapting and optimizing, the spin-coating technique to deposit lipids on a chemically etched Silicon (1,0,0) substrate, a morphological nanometer-scale characterization of the aforementioned samples has been provided. The AFM study showed that an initial layer of ordered vesicles is formed and, afterward, depending on details of the spin-coating preparation protocol and to the dimension of the silicon substrate, vesicle fusion and structural rearrangements of the lipid layers may occur.The present data disclose the possibility to control the lipid's structures by acting on spin-coating parameters with promising perspectives for novel applications of lipid films.     

Atomic Force Microscopy of Biological Membranes

Atomic force microscopy (AFM) is an ideal method to study the surface topography of biological membranes. It allows membranes that are adsorbed to flat solid supports to be raster-scanned in physiological solutions with an atomically sharp tip. Therefore, AFM is capable of observing biological molecular machines at work. In addition, the tip can be tethered to the end of a single membrane protein, and forces acting on the tip upon its retraction indicate barriers that occur during the process of protein unfolding. Here we discuss the fundamental limitations of AFM determined by the properties of cantilevers, present aspects of sample preparation, and review results achieved on reconstituted and native biological membranes.     

Atomic force microscopy: a powerful tool for high-resolution imaging of spermatozoa

Atomic force microscopy (AFM) has emerged as the only technique capable of real-time imaging of the surface of a living cell at nano-resolution. Since AFM provides the advantage of directly observing living biological cells in their native environment, this technique has found many applications in pharmacology, biotechnology, microbiology, structural and molecular biology, genetics and other biology-related fields. AFM has also proved to be a valuable tool for reproductive biologists. An exhaustive review on the various applications of AFM to sperm cells is presented. AFM has been extensively applied for determining the structural and topological features of spermatozoa. Unstained, unfixed spermatozoa in their natural physiological surroundings can be imaged by this technique which provides valuable information about the morphological and pathological defects in sperm cells as three-dimensional images with precise topographical details. Sperm head defects and the acrosome at the tip of the head responsible for fertilization, can be examined and correlated with the lack of functional integrity of the cell. Considerable amount of work is reported on the structural details of the highly condensed chromatin in sperm head using AFM. Detailed information on 3D topographical images of spermatozoa acquired by AFM is expected to provide a better understanding of various reproductive pathways which, in turn, can facilitate improved infertility management and/or contraceptive development.