Using carbon nanotube probes for high-resolution three-dimensional imaging of cells

While atomic force microscopy (AFM) has become a promising tool for visualizing membrane morphology of cells, many studies have reported the presence of artifacts such as cliffs on the edges of cells. These artifacts shield important structural features such as lamellopodia, filopodia, microvilli and membrane ridges, which represent characteristic status in signaling processes such as spreading and activation. These cliff-like edges arise from a premature contact of the probe side contact with the cell prior to the probe top apex-cell contact. Carbon nanotube (CNT) modified AFM probes were utilized to address this drawback. Using rat basophilic leukemia (RBL) cells, this work revealed that CNT probes diminish cliff-like artifacts and enabled visualization of entire membrane morphology and structural features in three dimensions. The high aspect ratio of CNT probes provides a very effective remedy to the cliff-like artifacts as well as tip convolution of conventional probes, which shall enhance the validity and application of AFM in cellular biology research.     

High-resolution imaging and nano-manipulation of biological structures on surface

In this mini-review we discuss our recent findings on imaging and manipulation of biological macromolecular structures by atomic force microscopy (AFM). In the first part of this review, we focus on high-resolution imaging of selected biological samples. AFM images of membrane proteins have revealed detailed conformational features related to identifiable biological functions. Different self-assembling behaviors of short peptides into supramolecular structures on various substrates under controlled environmental conditions have been systematically studied with AFM imaging. In the second part, we present a novel nano-manipulation technique for manipulating, isolating, amplifying, and sequencing of individual DNA molecules, which may find unique applications in the analysis of difficult sequence structures. Finally, we discuss how to characterize the elasticity of individual biomolecules and live cells. These results demonstrate that not only the high resolution capacity of the AFM is suited to resolve certain biological questions, but can also be applied to single molecule isolation and biomechanical analysis with its unique advantages.     

The structure of proteoglycan aggregate determined by atomic force microscopy

Proteoglycan aggregate is the major extracellular matrix component in cartilage, comprising about 18% of the dry weight of hyaline cartilage. The proteoglycan aggregate is the major substance in cartilage which resists compression in the joint. The purpose of this study was to utilize the newly developed imaging technique, Atomic force Microscopy (AFM), to visualize the ultrastructure of proteoglycan aggregates. The proteoglycan aggregate molecules were imaged in air using the tapping mode of the AFM. The images illustrated the ultrastructure of the aggregates, especially the individual proteoglycan and the core hyaluronic acid. In addition to the length and width of each molecule, the height of the proteoglycan aggregates and the individual proteoglycans could be directly measured. The images of the ultrastructures of proteoglycan aggregates visualized from the AFM are comparable with those using conventional electron microscopy approaches. Nevertheless, the sample preparation for AFM imaging does not involve fixation, staining, coating, and other routine procedures required for traditional electron microscopy imaging. Thus, this technique could be a simple alternative approach for future analysis of proteoglycan aggregate and its assembly.     

Time-lapse imaging of In Vitro myogenesis using atomic force microscopy

Myoblast therapy relies on the integration of skeletal muscle stem cells into distinct muscular compartments for the prevention of clinical conditions such as heart failure, or bladder dysfunction. Understanding the fundamentals of myogenesis is hence crucial for the success of these potential medical therapies. In this report, we followed the rearrangement of the surface membrane structure and the actin cytoskeletal organization in C2C12 myoblasts at different stages of myogenesis using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). AFM imaging of living myoblasts undergoing fusion unveiled that within minutes of making cell–cell contact, membrane tubules appear that unite the myoblasts and increase in girth as fusion proceeds. CLSM identified these membrane tubules as built on scaffolds of actin filaments that nucleate at points of contact between fusing myoblasts. In contrast, similarly behaving membrane tubules are absent during cytokinesis. The results from our study in combination with recent findings in literature further expand the understanding of the biochemical and membrane structural rearrangements involved in the two fundamental cellular processes of division and fusion.     

人脐静脉内皮细胞形貌与表面粘附力的原子力显微镜表征

目的:探讨原子力显微镜(AFM)在研究人脐静脉内皮细胞(ECV304)表面形貌、超微结构及纳米机械性质等方面的应用,讨论ECV304超微结构和机械性质与其功能的关系。方法:利用AFM对ECV304细胞的表面形貌及生物机械性质进行表征与测量。结果:在AFM下观察到用普通光学显微镜难以观察到的ECV304细胞的独特的形态结构,如细胞骨架、伪足及细胞边缘微丝等。ECV304细胞呈现长梭形、多角形、圆形等多种形态,细胞表面平均粗糙度为320.52±75.98 nm,表面均匀分布微绒毛,细胞周围有铺展的圆盘状物质。力曲线定量分析得出针尖与细胞表面的非特异性粘附力为75±14 pN。结论:通过AFM成像和力曲线测量表明,ECV304细胞呈圆形,多角形,梭形等多种形态,针尖与细胞膜表面问的粘附力比较小,约75±14pN。     

Atomic force microscopic imaging of 30 nm chromatin fiber from partially relaxed plant chromosomes

We have developed a procedure for partially relaxing the barley metaphase chromosomes and exposing fibrous structures from the chromosomes. The observation by atomic force microscopy (AFM) showed that the fibrous structures are typically 0.5 to 1 microm long and 40 to 50 nm in diameter. In higher magnification imaging, we found the fibrous structures were composed of aligned granules and looked like "knobby fiber." These observations are consistent with previously reported features of chromatin fiber observed by AFM and scanning electron microscopy, suggesting that the structures correspond to 30 nm chromatin fibers. We observed the chromatin fiber extending straight from the periphery of the chromosomes in most cases, but fibers with different shapes, such as loop and spiral, were also observed. The procedure reported here will provide a new approach for observing the organization of chromatin fiber to higher-order structures by AFM and other high-resolution microscopy.     

Visualization of cytoskeletal elements by the atomic force microscope

We describe a novel application of atomic force microscopy (AFM) to directly visualize cytoskeletal fibers in human foreskin epithelial cells. The nonionic detergent Triton X-100 in a low concentration was used to remove the membrane, soluble proteins, and organelles from the cell. The remaining cytoskeleton can then be directly visualized in either liquid or air-dried ambient conditions. These two types of scanning provide complimentary information. Scanning in liquid visualizes the surface filaments of the cytoskeleton, whereas scanning in air shows both the surface filaments and the total "volume" of the cytoskeletal fibers. The smallest fibers observed were ca. 50 nm in diameter. The lateral resolution of this technique was ca.20 nm, which can be increased to a single nanometer level by choosing sharper AFM tips. Because the AFM is a true 3D technique, we are able to quantify the observed cytoskeleton by its density and volume. The types of fibers can be identified by their size, similar to electron microscopy.     

Prototype cantilevers for quantitative lateral force microscopy

Prototype cantilevers are presented that enable quantitative surface force measurements using contact-mode atomic force microscopy (AFM). The "hammerhead" cantilevers facilitate precise optical lever system calibrations for cantilever flexure and torsion, enabling quantifiable adhesion measurements and friction measurements by lateral force microscopy (LFM). Critically, a single hammerhead cantilever of known flexural stiffness and probe length dimension can be used to perform both a system calibration as well as surface force measurements in situ, which greatly increases force measurement precision and accuracy. During LFM calibration mode, a hammerhead cantilever allows an optical lever "torque sensitivity" to be generated for the quantification of LFM friction forces. Precise calibrations were performed on two different AFM instruments, in which torque sensitivity values were specified with sub-percent relative uncertainty. To examine the potential for accurate lateral force measurements using the prototype cantilevers, finite element analysis predicted measurement errors of a few percent or less, which could be reduced via refinement of calibration methodology or cantilever design. The cantilevers are compatible with commercial AFM instrumentation and can be used for other AFM techniques such as contact imaging and dynamic mode measurements.     

3-D morphological characterization of the liver parenchyma by atomic force microscopy and by scanning electron microscopy

A comparative study of atomic force microscopy (AFM) and scanning electron microscopy (SEM) imaging of the healthy human liver parenchyma was carried out to determine the similarities and the differences. In this study, we compared the fine hepatic structures as observed by SEM and AFM. Although AFM revealed such typical hepatic structures as bile canaliculi and hepatocytes, it also showed the location of the nucleus and chromatin granules in rough relief structure, which was not visible by SEM. By contrast, SEM visualized other structures, such as microvilli, the central vein, and collagenous fibers, none of which was visualized by AFM. For better orientation and confirmation of most of the structures imaged by SEM and AFM, Congo Red-stained specimens were also examined. Amyloid deposits in the Disse's spaces were shown especially clearly in these images. The differences between the SEM and AFM images reflected the characteristics of the detection systems and methods used for sample preparation. Our results reveal that more detailed information on hepatic morphology is obtained by exploiting the advantages of both SEM and AFM.     

Visualization of single proteins from stripped native cell membranes: A protocol for high‐resolution atomic force microscopy

Atomic force microscopy (AFM) proved to be able to obtain high‐resolution three‐dimensional images of single‐membrane proteins, isolated, crystallized, or included in reconstructed model membranes. The extension of this technique to native systems, such as the protein immersed in a cell membrane, needs a careful manipulation of the biological sample to meet the experimental constraints for high‐resolution AFM imaging. In this article, a general protocol for sample preparation is presented, based on the mechanical stretch of the cell membrane. The effectiveness for AFM imaging has been tested on the basis of an integrated optical and AFM approach and the proposed method has been applied to cells expressing cystic fibrosis transmembrane conductance regulator, a channel involved in cystic fibrosis, showing the possibility to identify and analyze single proteins in the plasma membrane. Microsc. Res. Tech. 76:723–732, 2013. © 2013 Wiley Periodicals, Inc.     

Identification and ultrastructural imaging of photodynamic therapy-induced microfilaments by atomic force microscopy

Atomic force microscopy (AFM) is an emerging technique for imaging biological samples at subnanometer resolution; however, the method is not widely used for cell imaging because it is limited to analysis of surface topology. In this study, we demonstrate identification and ultrastructural imaging of microfilaments using new approaches based on AFM. Photodynamic therapy (PDT) with a new chlorin-based photosensitizer DH-II-24 induced cell shrinkage, membrane blebbing, and reorganization of cytoskeletons in bladder cancer J82 cells. We investigated cytoskeletal changes using confocal microscopy and atomic force microscopy. Extracellular filaments formed by PDT were analyzed with a tandem imaging approach based on confocal microscopy and atomic force microscopy. Ultrathin filaments that were not visible by confocal microscopy were identified as microfilaments by on-stage labeling/imaging using atomic force microscopy. Furthermore, ultrastructural imaging revealed that these microfilaments had a stranded helical structure. Thus, these new approaches were useful for ultrastructural imaging of microfilaments at the molecular level, and, moreover, they may help to overcome the current limitations of fluorescence-based microscopy and atomic force microscopy in cell imaging.     

Phase contrast and DIC illumination for AFM hybrids

High-resolution optical microscopy is an essential pre-requisite for life science force microscopy, particularly for applications in cell biology and medicine. Identification and validation of cells is typically established with techniques like phase contrast microscopy or differential interference contrast microscopy. The option to select or monitor individual cells online with such light microscopy techniques while performing atomic force microscopy (AFM) measurements is therefore extremely beneficial. Here, we report two conceptually different strategies to implement these light microscopy techniques in a fully functional AFM head at the ultimate resolution of the Abbe diffraction limit.     

AFM Feature Definition for Neural Cells on Nanofibrillar Tissue Scaffolds

Summary: A diagnostic approach is developed and implemented that provides clear feature definition in atomic force microscopy (AFM) images of neural cells on nanofibrillar tissue scaffolds. Because the cellular edges and processes are on the same order as the background nanofibers, this imaging situation presents a feature definition problem. The diagnostic approach is based on analysis of discrete Fourier transforms of standard AFM section measurements. The diagnostic conclusion that the combination of dynamic range enhancement with low‐frequency component suppression enhances feature definition is shown to be correct and to lead to clear‐featured images that could change previously held assumptions about the cell–cell interactions present. Clear feature definition of cells on scaffolds extends the usefulness of AFM imaging for use in regenerative medicine. SCANNING 34: 316–324, 2012. © 2012 Wiley Periodicals, Inc.     

Automated image analysis of atomic force microscopy images of rotavirus particles

A variety of biological samples can be imaged by the atomic force microscope (AFM) under environments that range from vacuum to ambient to liquid. Generally imaging is pursued to evaluate structural features of the sample or perhaps identify some structural changes in the sample that are induced by the investigator. In many cases, AFM images of sample features and induced structural changes are interpreted in general qualitative terms such as markedly smaller or larger, rougher, highly irregular, or smooth. Various manual tools can be used to analyze images and extract more quantitative data, but this is usually a cumbersome process. To facilitate quantitative AFM imaging, automated image analysis routines are being developed. Viral particles imaged in water were used as a test case to develop an algorithm that automatically extracts average dimensional information from a large set of individual particles. The extracted information allows statistical analyses of the dimensional characteristics of the particles and facilitates interpretation related to the binding of the particles to the surface. This algorithm is being extended for analysis of other biological samples and physical objects that are imaged by AFM.     

Visualization of vesicle transport along and between distinct pathways in neurites of living cells

Trafficking of secretory vesicles along neurites of PC12 cells was visualized by 2D and 3D real-time imaging using fluorescence microscopy. Vesicle motion along distinct pathways was directly seen. From an overlay of individual pathways, the underlying cytoskeletal filament could be imaged at a subwavelength resolution. Continuous vesicle transport was interrupted by periods of diffusive motion with concomitant pathway changes. Statistical analysis shows that such interruptions were distributed stochastically along the filament, indicating a limited processivity of motor proteins also in a cellular context. Periods of diffusive motion facilitated the interaction with actively transported vesicles. Frequent associations and dissociations of vesicles have been observed consistently, pointing to a functional relevance of vesicle cotransport.     

Atomic force microscopy imaging and 3-D reconstructions of serial thin sections of a single cell and its interior structures

The thin sectioning has been widely applied in electron microscopy (EM), and successfully used for an in situ observation of inner ultrastructure of cells. This powerful technique has recently been extended to the research field of atomic force microscopy (AFM). However, there have been no reports describing AFM imaging of serial thin sections and three-dimensional (3-D) reconstruction of cells and their inner structures. In the present study, we used AFM to scan serial thin sections approximately 60 nm thick of a mouse embryonic stem (ES) cell, and to observe the in situ inner ultrastructure including cell membrane, cytoplasm, mitochondria, nucleus membrane, and linear chromatin. The high-magnification AFM imaging of single mitochondria clearly demonstrated the outer membrane, inner boundary membrane and cristal membrane of mitochondria in the cellular compartment. Importantly, AFM imaging on six serial thin sections of a single mouse ES cell showed that mitochondria underwent sequential changes in the number, morphology and distribution. These nanoscale images allowed us to perform 3-D surface reconstruction of interested interior structures in cells. Based on the serial in situ images, 3-D models of morphological characteristics, numbers and distributions of interior structures of the single ES cells were validated and reconstructed. Our results suggest that the combined AFM and serial-thin-section technique is useful for the nanoscale imaging and 3-D reconstruction of single cells and their inner structures. This technique may facilitate studies of proliferating and differentiating stages of stem cells or somatic cells at a nanoscale.     

Imaging and Measuring the Molecular Force of Lymphoma Pathological Cells Using Atomic Force Microscopy

Atomic force microscopy (AFM) provides a new technology to visualize the cellular topography and quantify the molecular interactions at nanometer spatial resolution. In this work, AFM was used to image the cellular topography and measure the molecular force of pathological cells from B‐cell lymphoma patients. After the fluorescence staining, cancer cells were recognized by their special morphological features and then the detailed topography was visualized by AFM imaging. The AFM images showed that cancer cells were much rougher than healthy cells. CD20 is a surface marker of B cells and rituximab is a monoclonal antibody against CD20. To measure the CD20‐rituximab interaction forces, the polyethylene glycol (PEG) linker was used to link rituximab onto the AFM tip and the verification experiments of the functionalized probe indicated that rituximab molecules were successfully linked onto the AFM tip. The CD20‐rituximab interaction forces were measured on about 20 pathological cells and the force measurement results indicated the CD20‐rituximab binding forces were mainly in the range of 110–120 pN and 130–140 pN. These results can improve our understanding of the topography and molecular force of lymphoma pathological cells. SCANNING 35:40‐46, 2013. © 2012 Wiley Periodicals, Inc.     

Mechanical stimulation of individual stereocilia of living cochlear hair cells by atomic force microscopy

This paper describes the investigation of elastical properties and imaging of living cochlear hair bundles of inner (IHC) and outer hair cells (OHC) on the level of individual stereocilia. A custom-made AFM-setup was used, allowing to scan the mechano-sensitive structures of the inner ear under direct control of an upright differential interference contrast (DIC) microscope with a water-immersion objective. Scanning electron microscopy (SEM) images of the identical hair bundles obtained after AFM investigation demonstrated that forces up to 1.5 nanonewton (nN) did not cause obvious damage of the surface morphology of the stereocilia. These are the first images of hair bundles of living sensory cells of the organ of Corti by AFM. They display the tips of individual stereocilia and the typical V-shape of ciliary bundles. Since line scans clearly show that slope and force interaction depend on the elastical properties of stereocilia, quantitative stiffness measurements and stimulation of single transduction channels are suggested.     

光学原子力显微镜中的蒙特卡罗方法

扫描探针显微镜(Scanning probe microscopy,SPM)是显微镜的一个分支,它利用物理探针扫描标本形成样本表面图像.而原子力显微镜(Atomic force microscopy,AFM)是SPM中一种多功能的表面成像和测量工具,对导电、不导电、真空中、空气中或流体中的各种样本均可测量.原子力显微镜最面临的最大挑战之一是评估其在表面测量过程中所伴随的不确定度.本研究通过XYZ Phase的标定,对一台光学原子力显微镜进行了校准.该方法旨在克服在评估一些无法实验确定的不确定部件时遇到的困难,如尖端表面相互作用力和尖端几何.运用蒙特卡罗方法来确定根据相关容差和概率密度函数(PDFs)随机绘制参数而引起的相关不确定度.整个过程遵循《测量不确定度表示指南》(GUM)补编2.经本方法验证,原子力显微镜的评估不确定度为10nm左右.     

Dual resonance excitation system for the contact mode of atomic force microscopy

We propose an improved system that enables simultaneous excitation and measurements of at least two resonance frequency spectra of a vibrating atomic force microscopy (AFM) cantilever. With the dual resonance excitation system it is not only possible to excite the cantilever vibrations in different frequency ranges but also to control the excitation amplitude for the individual modes. This system can be used to excite the resonance frequencies of a cantilever that is either free of the tip-sample interactions or engaged in contact with the sample surface. The atomic force acoustic microscopy and principally similar methods utilize resonance frequencies of the AFM cantilever vibrating while in contact with the sample surface to determine its local elastic modulus. As such calculation demands values of at least two resonance frequencies, two or three subsequent measurements of the contact resonance spectra are necessary. Our approach shortens the measurement time by a factor of two and limits the influence of the AFM tip wear on the values of the tip-sample contact stiffness. In addition, it allows for in situ observation of processes transpiring within the AFM tip or the sample during non-elastic interaction, such as tip fracture.