肿瘤细胞的原子力显微术成像及表征研究

本文简单介绍了原子力显微镜的发展史,以及原子力显微镜的工作原理、工作模式、活细胞在生理状态下的成像方式等,特别介绍了生物型原子力显微镜、高速原子力显微镜在生物学领域的研究及应用。原子力显微镜在扫描速度、扫描范围、扫描精度方面的不断改进将为肿瘤细胞学研究提供源源不断的动力。本文着重阐述了原子力显微术在肿瘤领域的研究进展,包括原子力显微镜在肿瘤细胞形貌学特性、硬度、粘弹性方面的研究现状,并对原子力显微镜在肿瘤诊断及抗肿瘤药物研发方面的应用前景进行了展望。     

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.     

Atomic force microscopy of microvillous cell surface dynamics at fixed and living alveolar type II cells

Scanning probe techniques enable direct imaging of morphology changes associated with cellular processes at life specimen. Here, glutaraldehyde-fixed and living alveolar type II (ATII) cells were investigated by atomic force microscopy (AFM), and the obtained topographical data were correlated with results obtained by scanning electron microscopy (SEM) and confocal microscopy (CM). We show that low-force contact mode AFM at glutaraldehyde-fixed cells provides complementary results to SEM and CM. Both AFM and SEM images reveal fine structures at the surface of fixed cells, which indicate microvilli protrusions. If ATII cells were treated with Ca²⁺ channel modulators known to induce massive endocytosis, changes of the cell surface topography became evident by the depletion of microvilli. Low force contact mode AFM imaging at fixed ATII cells revealed a significant reduction of the surface roughness for capsazepine and 2-aminoethoxydiphenyl-borate (CPZ/2-APB)-treated cells compared to untreated control cells (Rc of 99.7 ± 6.8 nm vs. Rc of 71.9 ± 4.6 nm for N = 22), which was confirmed via SEM studies. CM of microvilli marker protein Ezrin revealed a cytoplasmic localization of Ezrin in CPZ/2-APB-treated cells, whereas a submembranous Ezrin localization was observed in control cells. Furthermore, in situ AFM investigations at living ATII cells using low force contact mode imaging revealed an apparent decrease in cell height of 17% during stimulation experiments. We conclude that a dynamic reorganization of the microvillous cell surface occurs in ATII cells at conditions of stimulated endocytosis.     

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.     

Investigations of local electrical surface characteristics by dynamical scanning force microscopy

A technique is presented that allows to obtain information about sample surface topography and local electrical surface properties simultaneously. A scanning electrical force microscope is used for that purpose which is based on an atomic force microscope (AFM) working in the dynamical mode. Different information channels contained in the cantilever excitation spectrum are separated by a lock-in technique. The physical content of the technique is discussed in detail and the influence of surface topography on the non-topographic imaging is demonstrated. Finally, the real advantages of cross-sectional sample preparation (as known from electron microscopy) for this kind of scanning probe microscopy with respect to various applications is presented.     

Quantitative Assessment of Tip Effects in Single-Molecule High-Speed Atomic Force Microscopy Using DNA Origami Substrates

High-speed atomic force microscopy (HS-AFM) is widely employed in the investigation of dynamic biomolecular processes at a single-molecule level. However, it remains an open and somewhat controversial question, how these processes are affected by the rapidly scanned AFM tip. While tip effects are commonly believed to be of minor importance in strongly binding systems, weaker interactions may significantly be disturbed. Herein, we quantitatively assess the role of tip effects in a strongly binding system using a DNA origami-based single-molecule assay. Despite its femtomolar dissociation constant, we find that HS-AFM imaging can disrupt monodentate binding of streptavidin (SAv) to biotin (Bt) even under gentle scanning conditions. To a lesser extent, this is also observed for the much stronger bidentate SAv–Bt complex. The presented DNA origami-based assay can be universally employed to quantify tip effects in strongly and weakly binding systems and to optimize the experimental settings for their reliable HS-AFM imaging.     

Application of integrated SECM ultra-micro-electrode and AFM force probe to biosensor surfaces

The integration of scanning electrochemical ultra-micro-electrode (UME) with atomic force microscope cantilever probe have been achieved by using a homemade photolithography system. A gold-film-coated AFM cantilever was insulated with photo resist coating and a pointed end of the AFM probe was opened by illuminating with maskless arbitrary optical micro-pattern generator. To realize precise control of probe sample distance constantly, the resulting scanning electrochemical microscopy (SECM)-AFM probe was operated using a dynamic force microscopy (DFM) technique with magnetic field excitation. From a steady-state voltammetric experiment, the effective electrode diameters of the probes thus prepared were estimated to be from 0.050 to 6.2 microm. The capability of this SECM-AFM probe have been tested using gold comb in the presence of Fe(CN)(6)(3-). The simultaneous imaging of the topography and electrochemical activity of the strip electrode was successfully obtained. We also used the SECM-AFM to examine in situ topography and enzymatic activity measurement. Comparison of topography and oxidation current profiles above enzyme-modified electrode showed active parts distribution of biosensor surface.     

Nanometer Scale Imaging and Biochemical Sensing

This seminar will cover two research topics in our group. The first one is nanometer scale sensing with living biological cells. In the biomedical sciences and technologies, the greatest advances in the last decade have been inspired by the genome project. What comes after the deciphering of the genetic code? Certainly one next step is the biochemistry driven by the gene, from the cellular nucleus to its organelles, cytoplasm and beyond. One important goal is to follow in real time the biochemical kinetics and dynamics of the living cell, much of which is in terms of small ions and biomolecules. Using both optical microscopy/spectroscopy and scanning probe microscopy, we have imaged single living cells and probed single molecule interactions.     

Dielectrophoretic force microscopy of aqueous interfaces

A novel scanning probe microscopy technique has allowed dielectrophoretic force imaging with nanoscale spatial resolution. Dielectrophoresis (DEP) traditionally describes the mobility of polarizable particles in inhomogeneous alternating current (ac) electric fields. Integrating DEP with atomic force microscopy allows for noncontact imaging with the image contrast related to the local electric polarizability. By tuning the ac frequency, dielectric spectroscopy can be performed at solid/liquid interfaces with high spatial resolution. In studies of cells, the frequency-dependent dielectrophoretic force is sensitive to biologically relevant electrical properties, including local membrane capacitance and ion mobility. Consequently, dielectrophoretic force microscopy is well suited for in vitro noncontact scanning probe microscopy of biological systems.     

Analysis of subcellular surface structure, function and dynamics

Analytics of single biological cells allows quantitative investigation from a structural, functional and dynamical point of view and opens novel possibilities to an unamplified subcellular analysis. In this article, we report on three different experimental methods and their applications to single cellular systems with a subcellular sensitivity down to the single molecule level. First, the subcellular surface structure of living bacteria (Corynebacterium glutamicum) was investigated with atomic force microscopy (AFM) at the resolution of individual surface layer (S-layer) proteins; discrimination of bacterial strains that lack the expression of hexagonally packed surface layer proteins was possible. Second, quantitative measurement of individual recognition events of membrane-bound receptors on living B-cells was achieved in single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy. And third, intracellular dynamics of translocating photoactivatable GFP in plant protoplasts (Nicotiana tabacum BY-2) was quantitatively monitored by two-photon laser scanning microscopy (2PLSM).     

Computer Simulation on TiO2 Nanostructure Films and Experimental Study Using Sol–Gel Method

Molecular dynamics simulation with an experimental work was performed on the TiO₂ nanostructure film. The Morse potential function was used for the interatomic interactions. Then, the equations of motion for molecules and atoms are solved by Verlet algorithm. The effects of deposition rate and the number of TiO₂ molecules were studied for morphology characterization of film surface. In addition, TiO₂ nanostructure film was prepared experimentally with the sol–gel dip-coating method. The results of MD simulations provide a reasonable compatibility with Dektak surface profiler, atomic force microscopy (AFM) and scanning electron microscopy (SEM) images due to the morphology and surface structure of films.     

Quantitative Assessment of Tip Effects in Single‐Molecule High‐Speed Atomic Force Microscopy Using DNA Origami Substrates

High‐speed atomic force microscopy (HS‐AFM) is widely employed in the investigation of dynamic biomolecular processes at a single‐molecule level. However, it remains an open and somewhat controversial question, how these processes are affected by the rapidly scanned AFM tip. While tip effects are commonly believed to be of minor importance in strongly binding systems, weaker interactions may significantly be disturbed. Herein, we quantitatively assess the role of tip effects in a strongly binding system using a DNA origami‐based single‐molecule assay. Despite its femtomolar dissociation constant, we find that HS‐AFM imaging can disrupt monodentate binding of streptavidin (SAv) to biotin (Bt) even under gentle scanning conditions. To a lesser extent, this is also observed for the much stronger bidentate SAv–Bt complex. The presented DNA origami‐based assay can be universally employed to quantify tip effects in strongly and weakly binding systems and to optimize the experimental settings for their reliable HS‐AFM imaging.     

A single-molecule view of conformational switching of DNA tethered to a gold electrode

Surfaces that can actively regulate binding affinities or catalytic properties in response to external stimuli are a powerful means to probe and control the dynamic interactions between the cell and its microenvironment. Active surfaces also enable novel functionalities in biosensors and biomolecular separation technologies. Although electrical stimuli are often appealing due to their speed and localization, the operation of these electrically activated surfaces has mostly been characterized with techniques averaging over many molecules. Without a molecular-scale understanding of how biomolecules respond to electric fields, achieving the ultimate detection sensitivity or localized biological perturbation with the ultimate resolution would be difficult. Using electrochemical atomic force microscopy, we are able to follow the conformational changes of individual, short DNA molecules tethered to a gold electrode in response to an applied potential. Our study reveals conformations and dynamics that are difficult to infer from ensemble measurements: defects in the self-assembled monolayer (SAM) significantly perturb conformations and adsorption/desorption kinetics of surface-tethered DNA; on the other hand, the SAM may be actively molded by the DNA at different potentials. These results underscore the importance of characterizing the systems at the relevant length scale in the development of electrically switchable biofunctional surfaces.     

Near-field scanning optical microscopy: a tool for nanometric exploration of biological membranes

Near-field scanning optical microscopy (NSOM) is an emerging optical technique that enables simultaneous high-resolution fluorescence and topography measurements. Here we discuss selected applications of NSOM to biological systems that help illustrate the utility of its high spatial resolution and simultaneous collection of both fluorescence and topography. For the biological sciences, these attributes seem particularly well suited for addressing ongoing issues in membrane organization, such as those regarding lipid rafts, and protein–protein interactions. Here we highlight a few NSOM measurements on model membranes, isolated biological membranes, and cultured cells that help illustrate some of these capabilities. We finish by highlighting nontraditional applications of NSOM that take advantage of the small probe to create nanometric sensors or new modes of imaging.     

Molecular dynamics simulation of surface morphology and thermodynamic properties of polyaniline nanostructured film

To develop predictive models in nanostructured films, there is an ongoing research to validate molecular dynamics (MD) simulation results with experimental data. The morphology and surface topography of polyaniline (PANI) nanostructured film coated on a TiO₂ nanocrystalline surface were investigated by scanning electron microscopy and atomic force microscopy, respectively. The atomistic model of the simulated PANI was generated using energy minimization with a condensed‐phase optimized molecular potential for atomistic studies force field function to reach a thermodynamic equilibrium state. Various parameters of PANI such as density, energy, cavity size, and free volume distributions are calculated. MD simulation has also been used to obtain specific volume (V) as a function of temperature (T). It is demonstrated that this V–T curve can be used to determinate glass transition temperature T_g, reliably. Although experimental data available for the PANI film are very limited, simulation results such as density and T_g are in good agreement with the experimental values reported in the literature. Comparison of the surface topography of PANI demonstrates a reasonable trend between atomic force microscopy image analysis and the MD simulation results at various temperatures. Copyright © 2014 John Wiley & Sons, Ltd.     

Nanosensing of Fcγ receptors on macrophages

Determining the distribution of specific binding sites on biological samples with high spatial accuracy (in the order of several nanometer) is an important challenge in many fields of biological science. Combination of high-resolution atomic force microscope (AFM) topography imaging with single-molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events. The identification and localization of specific receptor binding sites on complex heterogeneous biosurfaces such as cells and membranes are of particular interest in this context. Simultaneous topography and recognition imaging was used to unravel the nanolandscape of cells of the immune system such as macrophages. The most studied phagocytic receptors include the Fc receptors that bind to the Fc portion of immunoglobulins. Here, nanomapping of FcγRs (Fc receptors for immunoglobulin G (IgG)) was performed on fixed J774.A1 mouse macrophage cell surfaces with magnetically coated AFM tips functionalized with Fc fragments of mouse IgG via long and flexible poly(ethylene glycol) linkers. Because of possible AFM tip engulfment on living macrophages, appropriate cell fixation procedure leaving the binding activity of FcγRs practically intact was elaborated. The recognition maps revealed prominent spots (microdomains) more or less homogeneously distributed on the macrophage surface with the sizes from 4 to 300 nm. Typical recognition image contained about ∼4% of large clusters (>200 nm), which were surrounded by a massive number (∼50%) of small-size (4–30 nm) and the rest by middle-size (50, 150 nm) domains. These spots were detected from the decrease of oscillation amplitude during specific binding between Fc-coated tip and FcγRs on macrophage surfaces. In addition, the effect of osmotic swelling on the topographical landscape of macrophage surfaces and on the reorganization of FcγRs was investigated.     

扫描离子电导显微镜的原理及应用

扫描离子电导显微镜(SICM)是一种扫描探针显微技术,通过测定超微玻璃管探针的离子电流,它能够非接触地扫描样品表面,进而研究样品的形貌及性质。SICM具有成像分辨率高、探针易于制备和对被成像物体无损伤等特点,特别适用于研究生理条件下的活体细胞,是一种与扫描电化学显微镜及原子力显微镜互补的扫描探针显微镜技术。SICM能够对软界面及表面,如活细胞表面的显微结构,进行高分辨率成像;并能够与其它技术联用,研究细胞形貌与功能的关系;还能控制沉积特定分子,实现纳米尺度的显微操作与加工。本文对SICM的发展历史、仪器构造、基本原理及应用进行了综述。     

基于AFM的药物刺激前后淋巴瘤活细胞的形貌及弹性的变化

原子力显微镜(AFM)的发明为研究单个活细胞的形貌结构及物理特性提供了新的技术手段.然而,由于缺少合适的固定方法,利用AFM对动物悬浮活细胞的形貌进行高分辨率成像还面临着巨大的挑战.本文提出一种基于微柱阵列和静电吸附相结合的动物悬浮细胞固定方法.通过微柱阵列的机械钳制和多聚赖氨酸的静电吸附实现了对单个淋巴瘤B细胞的固定,并在此基础上利用AFM动态观测了不同浓度Rituximab刺激下淋巴瘤B细胞的表面形貌及弹性的变化.经过0.2 mg·mL-1的Rituximab刺激2 h后,细胞表面的褶皱增加,细胞的杨氏模量从196 kPa减小到183 kPa.经过0.5 mg·mL-1的Rituximab刺激2 h后,细胞形貌发生显著变化并出现突起结构,细胞的杨氏模量从234 kPa减小到175 kPa.实验结果表明淋巴瘤细胞形貌和弹性变化的幅度随着Rituximab刺激浓度的增加而增加,加深了对Rituximab作用效果的认识.     

Deciphering the nanometer-scale organization and assembly of Lactobacillus rhamnosus GG pili using atomic force microscopy

In living cells, sophisticated functional interfaces are generated through the self-assembly of bioactive building blocks. Prominent examples of such biofunctional surfaces are bacterial nanostructures referred to as pili. Although these proteinaceous filaments exhibit remarkable structure and functions, their potential to design bioinspired self-assembled systems has been overlooked. Here, we used atomic force microscopy (AFM) to explore the supramolecular organization and self-assembly of pili from the Gram-positive probiotic bacterium Lactobacillus rhamnosus GG (LGG). High-resolution AFM imaging of cell preparations adsorbed on mica revealed pili not only all around the cells, but also in the form of remarkable star-like structures assembled on the mica surface. Next, we showed that two-step centrifugation is a simple procedure to separate large amounts of pili, even though through their synthesis they are covalently anchored to the cell wall. We also found that the centrifuged pili assemble as long bundles. We suggest that these bundles originate from a complex interplay of mechanical effects (centrifugal force) and biomolecular interactions involving the SpaC cell adhesion pilin subunit (lectin-glycan bonds, hydrophobic bonds). Supporting this view, we found that pili isolated from an LGG mutant lacking hydrophilic exopolysaccharides show an increased tendency to form tight bundles. These experiments demonstrate that AFM is a powerful platform for visualizing individual pili on bacterial surfaces and for unravelling their two-dimensional assembly on solid surfaces. Our data suggest that bacterial pili may provide a generic approach in nanobiotechnology for elaborating functional supramolecular interfaces assembled from bioactive building blocks.     

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。