Atomic force microscopy of native human metaphase chromosomes in a liquid

The present study introduces a method for obtaining three-dimensional images of native (i.e., unfixed) chromosomes by atomic force microscopy (AFM) in a liquid. Human metaphase chromosomes were isolated from a human lymphoblast-like cell line, K562, by the hexylene glycol procedure according to Wray and Stubble- field (1970), adsorbed on a silane-coated glass slide, and observed in a dynamic force mode (i.e., intermittent contact mode) of AFM in a hexylene buffer solution. In adequate operating conditions, the shape of chromosomes with paired chromatids was clearly and three-dimensionally observed by AFM. At high magnification, globular or fibrous structures about 50 nm thick could be found on the surface of each chromaid, implying that chromatin fibers were strongly wound or twisted in the chromatid. Thus, AFM imaging enabled the direct visualization of native chromosomes in a liquid at high resolution--which is comparable with that of scanning electron microscopy--and can serve to analyze the mechanism of chromosome condensation and separation in relation to the structure of chromosomes.     

Imaging and manipulating chromosomes with the atomic force microscope

Polytene chromosomes from the salivary gland cells of Drosophila melanogaster were examined by atomic force microscopy. The atomic force microscope (AFM) was capable of resolving chromosomal features down to the limits of the tip sharpness, about 500 Å for pyramidal-shaped tips. Resolution was increased to 300 Å by using electron beam deposited (EBD) tips with high aspect ratios. This significantly exceeds the resolution obtainable with conventional optical microscopes, but at the cost of compromising the structural integrity of the sample. A reasonable compromise was achieved by using oxide-sharpened tips. In this case high resolution was obtained without sample degradation, but when desired these tips were also capable of sample disintegration with increased scanning force and rate. Thus, oxide-sharpened tips were used to precisely dissect defined chromosomal regions to illustrate their potential use in genetic mapping efforts. This study illustrates the utility of the AFM in the characterization and manipulation of chromosomes and chromosomal DNA.     

Nonidet P-40致流感病毒脱包膜的AFM观察研究

目的 利用原子力显微镜(AFM)观察经非离子表面活性剂Nonidet P-40(NP-40)不同浓度系列处理的A型流感病毒表面形态变化,观察不同表面活性剂-病毒表面相互作用情况,以提供一种较为温和的病毒表面裂解条件,为利用AFM进一步研究病毒下层结构提供基础.方法 用不同浓度的非离子犁表面活性剂NP-40对完整的A型流感病毒进行处理,以轻敲模式经AFM成像,获得病毒球状体和丝状体的高度图、振幅图以及相位图,并观察和比较不同浓度非离子表面活性剂对病毒表面形态和结构的影响.结果 NP-40各浓度对病度表面破坏程度不一,病毒随NP-40浓度增高而逐渐降解,并出现部分剥离病毒表面.暴露下层衣壳,更清晰地展示包膜下层表面突起的表面形态学结果.结论 通过表面活性剂优化处理病毒颗粒,实现了利用AFM观测流感病毒包膜下形态结构的设想.     

Single-molecule anatomy by atomic force microscopy and recognition imaging

Atomic force microscopy (AFM) has been a useful technique to visualize cellular and molecular structures at single-molecule resolution. The combination of imaging and force modes has also allowed the characterization of physical properties of biological macromolecules in relation to their structures. Furthermore, recognition imaging, which is obtained under the TREC(TM) (Topography and RECognition) mode of AFM, can map a specific protein of interest within an AFM image. In this study, we first demonstrated structural properties of purified α Actinin-4 by conventional AFM. Since this molecule is an actin binding protein that cross-bridges actin filaments and anchors it to integrin via tailin-vinculin-α actinin adaptor-interaction, we investigated their structural properties using the recognition mode of AFM. For this purpose, we attached an anti-α Actinin-4 monoclonal antibody to the AFM cantilever and performed recognition imaging against α Actinin-4. We finally succeeded in mapping the epitopic region within the α Actinin-4 molecule. Thus, recognition imaging using an antibody coupled AFM cantilever will be useful for single-molecule anatomy of biological macromolecules and structures.     

狂犬病病毒CTN-1v株原子力显微镜观察结果的初步分析

目的 利用原子力显微镜对狂犬病病毒进行观察.方法 超速离心制备狂犬病病毒CTN-1v株,采用磷钨酸负染透射电子显微镜进行观察,在此基础上进行原子力显微镜观察,采用轻敲模式在大气常温下扫描成像.结果 透射电子显微镜观察到狂犬病病毒的典型弹状病毒粒子,透射电镜提供病毒二维图像,可见刺突结构,原子力显微镜则呈现了狂犬病病毒三维图像,且可见病毒表面有凹凸不平的特征和边缘有齿轮状的突起,同时获得表面粗糙度等可以量化指标.两种方法最终得到相似的形态学结果.结论 利用原子力显微镜首次观察到狂犬病病毒的三维形态结构,与透射电镜观察相比,原子力显微镜是一种制样简单、观察直观的新型病毒形态学研究工具.

Abstract：

Objective To explore the application of atomic force microscopy( AFM ) on the research of morphology of the rabies viruses. Methods To prepare the rabies virus CTN-1v strains by ultracentrifugation, and observe it with transmission electron microscopy (TEM) which negatively stained by phosphotungstic acid. Then study the morphology of rabies virus with AFM based on the result of TEM. AFM image applies the tapping mode to rabies virus without any further treatment in air at room temperature. Results The TEM image is two-dimensional image which can be seen the classical bullet-shaped structure,and the spike structure can also be seen. The AFM image showed the rabies virus morphology with three-dimensional image which can shows the characteristics of the virus surface and edge. The rabies virus particle was successfully observed by TEM or AFM methods. Conclusion It's the first time to get the three-dimensional morphological structure of rabies virus by atomic force microscopy, compared with transmission electron microscopy, AFM is a new research tool for viral morphology study with the advantages of simple sample preparing and intuitionistic and visible interface for researchers.     

Dynamic mechanical analysis of collagen fibrils at the nanoscale

Low frequency (0.1-2 Hz) dynamic mechanical analysis on individual type I collagen fibrils has been carried out using atomic force microscopy (AFM). Both the elastic (static) and viscous (dynamic) responses are correlated to the characteristic axial banding, gap and overlap regions. The elastic modulus (～5 GPa) on the overlap region, where the density of tropocollagen is highest, is 160% that of the gap region. The amount of dissipation on each region is frequency dependent, with the gap region dissipating most energy at the lowest frequencies (0.1 Hz) and crossing over with the overlap region at ～0.75 Hz. This may reflect an ability of collagen fibrils to absorb energy over a range of frequencies using more than one mechanism, which is suggested as an evolutionary driver for the mechanical role of type I collagen in connective tissues and organs.     

Scanning near field optical/atomic force microscopy of bromodeoxyuridine-incorporated human chromosomes

The present study applied scanning near field optical/atomic force microscopy (SNOM/AFM) to the observation of human chromosomes immunostained with an anti-BrdU antibody after incorporation of BrdU into DNA. Human lymphocytes were cultured in BrdU for 72 h and their chromosomes were prepared with a standard method for light microscopy. After additional fixation with 15% formalin in phosphate buffered saline, the specimens were denatured with 2N HCI with 0.1% Triton-X 100, immunostained with the anti-BrdU antibody, and observed both by fluorescence microscopy and by SNOM/AFM. The preparation technique used in the present study enabled the differential staining of sister chromatids in each chromosome, and sister chromatid exchanges (SCEs) were recognized in some chromosomes of the metaphase spread. Observations of the specimens by SNOM/AFM further provided the simultaneous collection of topographical and fluorescent images of the same portions of BrdU-incorporated chromosomes. The resolution of the fluorescence images by SNOM/AFM was greater than that obtained by fluorescence microscopy. Superimposition of topographical and fluorescent images of the chromosomes is useful for the precise analysis of the fine structure of chromosomes in relation to the SCEs. The application of SNOM/AFM to the BrdU-incorporated chromosomes is thus useful for the analysis of the fine structure of chromosomes in relation to their function.     

Total internal reflection microscopy and atomic force microscopy (TIRFM-AFM) to study stress transduction mechanisms in endothelial cells

The cytoskeleton plays a key role in providing strength and structure to the cell. A force balance exists between the cytoskeleton and the extracellular matrix/substratum via the focal contact regions. The purpose of this study is to integrate atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) data to determine the effect of localized force application over the cell surface on the cell's focal contacts size and position. TIRFM gives detailed information on the cell-substrate contact regions and AFM is a tool for elasticity measurements, force application, and topographic surface mapping of the cell. TIRFM data were calibrated by varying the intensity of the evanescent wave to change the interfacial angle at the glass-cell interface. The individual focal contact intensity was found to decrease with increasing interfacial angles from 66 degrees to 80 degrees as the depth of penetration varied from 150 to 66 nm. A measure of cellular mechanical properties was obtained by collecting a set of force curves over the entire cell using the Bioscope AFM. The nuclear region appears to be stiffer than the cell body. Preliminary results of the nanonewtons force application to the cell surface indicate that the cell-substrate contacts rearrange to offset the force. It is evident that the stress applied to the surface is transmitted to the cell-substrate contact region.     

Dynamics of hydrated polyurethane biomaterials: Surface microphase restructuring,protein activity and platelet adhesion

Microphase separation is a central feature of segmented polyurethane biomaterials and contributes to the biological response to these materials. In this study we utilized atomic force microscopy (AFM) to study the dynamic restructuring of three polyurethanes having soft segment chemistries of interest in biomedical applications. For each of the materials we followed the changes in near surface mechanical properties during hydration, as well as fibrinogen activity and platelet adhesion on these surfaces. Both AFM phase imaging and force mode analysis demonstrated that these polyurethane biomaterials underwent reorientation and rearrangement resulting in a net enrichment of hard domains at the surface. Fibrinogen activity and platelet adhesion on the polyurethane surfaces were found to decrease with increasing hydration time. The findings suggest that water-induced enrichment of hydrophilic hard domains at the surface changes the local surface physical and chemical properties in a way that influences the conformation of fibrinogen, changing the availability of the platelet-binding sites in the protein. This work demonstrates that the hydrated polyurethane biomaterial interface is a complex and dynamic environment where the surface chemistry is changing, altering the activity of fibrinogen and affecting blood platelet adhesion.     

Imaging of chromosomes at nano-meter scale resolution using scanning near-field optical/atomic force microscopy

Topographic and fluorescent images of whole barley chromosomes stained with YOYO-1 were observed simultaneously by scanning near-field optical/ atomic force microscopy (SNOM/AFM). The chromosome was relatively smooth and flat in the topographic images and no significant difference in height was present between regions of high fluorescent and low fluorescent intensity in the chromosomes. The telomeric region, labeled by fluorescence in situ hybridization (FISH) method, was also observed by SNOM/AFM at high resolution, and fluorescent signals of the telomeric region were clearly defined on the topographic image of chromatin fibers on the chromosome at the nano-meter scale level. Although the telomeric signals were usually visualized as a single fluorescent region at the end of sister chromatids by conventional light microscopy, they were observed separately as two fluorescent regions, less than 100-200 nm distance, using the SNOM/AFM. The SNOM/AFM offers great potential in identifying particular single gene location on chromosomes in the near future.     

The structure of C-banded human metaphase chromosomes as observed by atomic force microscopy

The ultrastructure of C-banded human metaphase chromosomes was studied by the combined use of light microscopy and atomic force microscopy (AFM). Light microscopy of the C-banded chromosomes showed that the centromeric regions of all chromosomes except the Y chromosome were positively stained. AFM further revealed that the C-positive region was higher than the C-negative region. The area of the C-positive region was specific depending on each chromosome; it ranged from the centromere to the proximal end of the long arm in chromosome 1, while it was restricted to the centromere in chromosomes 2 and 3. At higher magnification, chromatin fibers about 50 nm thick were clearly shown in the entire length of the chromosomes. In the C-positive region, these chromatin fibers were densely packed, while chromatin fibers were loosely packed with gentle twisting in the C-negative region. These AFM findings suggest that certain factors related to the chromatin fiber compaction remain in the C-positive region even after successive C-banging treatment.     

High-speed AFM and nano-visualization of biomolecular processes

Conventional atomic force microscopes (AFMs) take at least 30–60 s to capture an image, while dynamic biomolecular processes occur on a millisecond timescale or less. To narrow this large difference in timescale, various studies have been carried out in the past decade. These efforts have led to a maximum imaging rate of 30–60 ms/frame for a scan range of ~250 nm, with a weak tip–sample interaction force being maintained. Recent imaging studies using high-speed AFM with this capacity have shown that this new microscope can provide straightforward and prompt answers to how and what structural changes progress while individual biomolecules are at work. This article first compares high-speed AFM with its competitor (single-molecule fluorescence microscopy) on various aspects and then describes high-speed AFM instrumentation and imaging studies on biomolecular processes. The article concludes by discussing the future prospects of this cutting-edge microscopy.     

Development of a nano manipulator based on an atomic force microscope coupled with a haptic device: a novel manipulation tool for scanning electron microscopy

We developed a novel nano manipulator based on an atomic force microscope (AFM) that can be operated inside the sample chamber of a scanning electron microscope (SEM). This AFM manipulator is also coupled with a haptic device, and the nanometer-scale movement of the AFM cantilever can be scaled up to the millimeter-scale movement of the pen handle of the haptic device. Using this AFM manipulation system, we were able to observe the AFM cantilever and samples under the SEM and obtain topographical images of the AFM under the SEM. These AFM images contained quantitative height information of the sample that is difficult to obtain from SEM images. Our system was also useful for positioning the cantilever for accurate AFM manipulation because the manipulation scene could be directly observed in real time by SEM. Coupling of the AFM manipulator with the haptic device was also useful for manipulation in the SEM since the operator can move the AFM probe freely at any position on the sample surface while feeling the interaction force between the probe and the sample surface. We tested two types of cutting methods: simple cutting and vibration cutting. Our results showed that vibration cutting with probe oscillation is very useful for the dissection of biological samples which were dried for SEM observation. Thus, cultivated HeLa cells were successfully micro-dissected by vibration cutting, and the dissection process could be observed in real time in the SEM. This AFM manipulation system is expected to serve as a powerful tool for dissecting various biological samples at the micro and nanometer-scale under SEM observation.     

The measurement of biomechanical properties of porcine articular cartilage using atomic force microscopy

We have recently demonstrated that indentation-type atomic force microscopy (IT-AFM) is capable of detecting early onset osteoarthritis (OA) (Stolz, 2009). This study was based on biopsies, using a desk-top commercial atomic force microscope (AFM). However, cartilage analysis in the knee joints needs to be non-destructive to avoid new seeding points for OA by the taking of biopsies. This requires bringing the probe tip in contact with the articular cartilage (AC) surface inside the joint. Here we present our recent progress towards a medical instrument for performing such IT-AFM measurements for in-vivo knee diagnostics. The scanning force arthroscope (SFA) integrates a miniaturized AFM into a standard arthroscopic sleeve, and is used for direct, quantitative, in situ inspection of AC (Imer et al., 2006). The stabilization and the positioning of the instrument relative to the surface under investigation were performed by means of eight inflatable balloons. An integrated three-dimensional, piezoelectric scanner allowed raster scanning and probing of a small area of cartilage around the point of insertion. An AFM probe with an integrated deflection sensor was mounted at the distal end of the instrument. Using this instrument, several measurements were performed on agarose gel and on porcine cartilage samples. The load-displacement curves obtained were analyzed and the dynamic elastic moduli | E(*) | were calculated. A good correlation between these values and those published in the scientific literature was found. Therefore, we concluded that the SFA can provide quantitative measurements to detect early pathological changes in OA.     

Nuclear pore function viewed with atomic force microscopy

In this review we focus on studies using atomic force microscopy (AFM) to describe the function of nuclear pore complexes (NPC). After a short introduction of AFM we follow the route of cargo molecules from the cytosol into the nucleus. AFM visualizes cargo before translocation into the nucleoplasm, cargo docking at the cytoplasmic NPC surface, cargo passing through the NPC and changes in NPC conformation in response to ATP, Calcium and pH. We discuss AFM experiments on nuclear envelopes on the basis of previous data obtained with more conventional techniques such as electron microscopy, confocal microscopy and other imaging techniques. Finally we draw attention to the recently developed nuclear hourglass technique that serves as a new electrophysiological approach to studying the structure-function relationship of NPC in combination with AFM at a molecular level.     

Encapsulated cells: an atomic force microscopy study

Two types of cells--human platelets and spore cells--were encapsulated in polymer shells by adsorbing polyanions and polycations in a stepwise fashion. The encapsulated cells were attached to gold and silicon surfaces and their morphological and adhesion properties were studied in air using tapping mode atomic force microscopy (AFM). The roughness of the encapsulated cells increased upon the addition of a new polymer layer. The increase in roughness can be attributed to the formation of a shell around the cells, which is stabilized by electrostatic interactions, as well as to the drying effects associated with the immobilization and dehydration of the cells. Trigger mode was used to perform the force imaging and map out the adhesion characteristics of the cells. Systematic "maps" of the adhesion properties of the encapsulated cells to clean and amine terminated AFM tips were collected. The adhesion force data for the different tips and encapsulated cells showed dependence not only on the number and thickness of the polymer layers, but also on the interactions between these layers. The encapsulated cells' morphology and roughness characteristics remained intact over a substantial storage period. This stability and adhesion properties make them suitable building blocks for the design and construction of biomimetic templates where AFM is used as the primary tool to do the fabrication.     

Effects of surface undulations of biphasic calcium phosphate tablets on human osteoblast behavior

In this work, the in vitro behavior of human osteoblast cells on the undulated surfaces of biphasic calcium phosphate tablets was investigated. The tablets were produced by uniaxial pressing with convex cylindrical undulations occupying only half of the surface area; the other half was flat. Chemical and physical characterization was performed by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). XRD and FTIR analyses revealed the presence of hydroxyapatite (HA) and alpha-tricalcium phosphate (alpha-TCP) in a well-defined ratio. Moreover, microtopography, evaluated by SEM and AFM, was similar on the flat region and on that with undulations. However, surface undulations induced different cellular arrangements, confirming the influence of the macrotopography on the cells orientation.     

碳纳米管原子力显微镜探针对生物样品高分辨率成像的研究

本研究将碳纳米管安装到原子力显微镜的标准硅探针上 ,制备了碳纳米管原子力显微镜针尖 ,运用其对生物样品进行高分辨率的成像研究 ,成功地获得了DNA的精细结构和G型免疫球蛋白 (Immunoglobulin G ,IgG)的Y形结构 ,这用传统的原子力显微镜针尖是无法获得的     

Imaging the lamellipodium of migrating epithelial cells in vivo by atomic force microscopy

Cell locomotion originates at a specific region of the cell surface, the leading edge of a migrating cell. Various factors have been proposed to contribute to the propulsion of a cell over the substratum. Rapid turnover processes of cytoskeletal elements inside the cell and insertion of new plasma membrane at the leading edge of the cell permit the extension of a cell in a given direction. Our goal was to image in vivo plasma membrane turnover by means of atomic force microscopy (AFM) and to resolve dynamic processes at the nanometer level. As an experimental model we used migrating kidney cells derived from the Madin-Darby canine kidney (MDCK) cell line that was transformed by alkaline stress. These so-called MDCK-F cells exhibit spontaneous calcium-dependent oscillatory activity of plasma membrane potential associated with cell locomotion. We imaged cells during migration and observed dynamic invagination processes in the cell surface close to the leading edge, indicating internalization of plasma membrane. Invaginations were prevented by removal of calcium from the perfusate. During calcium reduction plasma membrane uncoupled from the underlying cytoskeleton and lipidic pores with diameters of about 30 nm could be disclosed and imaged. This study demonstrates that the AFM can readily trace dynamic physiological processes in vivo, emphasizing the potential role of calcium in maintaining plasma membrane integrity and function.     

Precise positioning of cancerous cells on PDMS substrates with gradients of elasticity

In this work the novel method to create PDMS substrates with continuous and discrete elasticity gradients of different shapes and dimensions over the large areas was introduced. Elastic properties of the sample were traced using force spectroscopy (FS) and quantitative imaging (QI) mode of atomic force microscopy (AFM). Then, fluorescence microscopy was applied to investigate the effect of elastic properties on proliferation of bladder cancer cells (HCV29). Obtained results show that cancerous cells proliferate significantly more effective on soft PDMS, whereas the stiff one is almost cell-repellant. This strong impact of substrate elasticity on cellular behavior is driving force enabling precise positioning of cells.