原子力显微镜 (AFM) 在细菌生物被膜研究中的应用

原子力显微镜(AFM)作为一项重要的表面可视化技术,以其独特的优势(纳米级的空间分辨率、皮牛级力灵敏度、免标记、可在溶液环境下工作)被广泛应用于生物被膜的研究。AFM不仅可以在近生理环境下对生物被膜表面超微形貌进行可视化表征,同时还可以通过纳米压痕对生物被膜的机械特性(弹性和粘性)进行定量测量,利用AFM单细胞和单分子力谱技术可以获得生物被膜形成过程中细胞-基底以及细胞-细胞之间的相互作用力,为生物被膜的实时原位系统研究提供了可行性。本文简述了AFM的基本操作原理,综述了近年来AFM用于生物被膜表面超微结构成像、机械特性测量以及相互作用力研究方面的进展,并对AFM在生物被膜研究中面临的问题和未来的发展方向进行了讨论。     

Ultrastable atomic force microscopy: Improved force and positional stability

Atomic force microscopy (AFM) is an exciting technique for biophysical studies of single molecules, but its usefulness is limited by instrumental drift. We dramatically reduced positional drift by adding two lasers to track and thereby actively stabilize the tip and the surface. These lasers also enabled label-free optical images that were spatially aligned to the tip position. Finally, sub-pN force stability over 100 s was achieved by removing the gold coating from soft cantilevers. These enhancements to AFM instrumentation can immediately benefit research in biophysics and nanoscience.     

Ultra-high resolution imaging of DNA and nucleosomes using non-contact atomic force microscopy

Visualisation of nano-scale biomolecules aids understanding and development in molecular biology and nanotechnology. Detailed structure of nucleosomes adsorbed to mica has been captured in the absence of chemical-anchoring techniques, demonstrating the usefulness of non-contact atomic force microscopy (NC-AFM) for ultra-high resolution biomolecular imaging. NC-AFM offers significant advantages in terms of resolution, speed and ease of sample preparation when compared to techniques such as cryo-electron microscopy and X-ray crystallography. In the absence of chemical modification, detailed structure of DNA deposited on a gold substrate was observed for the first time using NC-AFM, opening up possibilities for investigating the electrical properties of unmodified DNA.     

Direct visualization of KirBac3.1 potassium channel gating by atomic force microscopy

KirBac3.1 belongs to a family of transmembrane potassium (K⁺) channels that permit the selective flow of K-ions across biological membranes and thereby regulate cell excitability. They are crucial for a wide range of biological processes and mutations in their genes cause multiple human diseases. Opening and closing (gating) of Kir channels may occur spontaneously but is modulated by numerous intracellular ligands that bind to the channel itself. These include lipids (such as PIP₂), G-proteins, nucleotides (such as ATP) and ions (e.g. H⁺, Mg²⁺, Ca²⁺). We have used high-resolution atomic force microscopy (AFM) to examine KirBac3.1 in two different configurations. AFM imaging of the cytoplasmic surface of KirBac3.1 embedded in a lipid bilayer has allowed visualization of the tetrameric assembly of the ligand-binding domain. In the absence of Mg²⁺, the four subunits appeared as four protrusions surrounding a central depression corresponding to the cytoplasmic pore. They did not display 4-fold symmetry, but formed a dimer-of-dimers with 2-fold symmetry. Upon addition of Mg²⁺, a marked rearrangement of the intracellular ligand-binding domains was observed: the four protrusions condensed into a single protrusion per tetramer, and there was an accompanying increase in protrusion height. The central cavity within the four intracellular domains also disappeared on addition of Mg²⁺, indicating constriction of the cytoplasmic pore. These structural changes are likely transduced to the transmembrane helices, which gate the K⁺ channel. This is the first time AFM has been used as an interactive tool to study K⁺ channels. It has enabled us to directly measure the conformational changes in the protein surface produced by ligand binding.     

Visualization of chromatin folding patterns in chicken erythrocytes by atomic force microscopy （AFM）

The organization of the higher order structure of chromatin in chicken erythrocytes has been examined with tapping-mode scanning force microscopy under conditions close to their native envirinment.Reproducible highresolution AFM images of chromatin compaction at several levels can be demonstrated.An extended beads-on-astring (width of - 15-20nm,height of - 2-3nm for each individual nucleosome) can be consistently observed.Furthermore,superbeade (width of - 40nm,height of - 7nm) are demonstrated.Visualization of the solenoid conformation at the level of 30nm chromatin fiber is attained either by using AFM or by using electron microscopy.In addition,tightly coiled chromatin fibers (- 50-60nm and - 90-110nm) can be revealed.Our data suggest that the chromatin in the interphase nucleus of chicken erythrocyte represents a high-order conformation and AFM provides useful high-resolution structural information concerning the folding pattern of interphase chromatin fibers.     

High-speed atomic force microscopy: Imaging and force spectroscopy

Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.     

女贞和珊瑚树叶片表面特征的AFM观察

应用原子力显微镜观察了女贞(Ligustrum lucidum)、珊瑚树(Viburnum odoratissimum)幼叶和成熟叶的表面特征,并探讨了叶面微结构对滞尘能力的可能影响以及抵抗干旱、污染物等胁迫的能力。女贞幼叶和成熟叶正背面的粗糙度Ra分别为417.8、794.5,1069、957.4 nm;珊瑚树幼叶和成熟叶正背面的粗糙度Ra分别为471.3、469.6,291.1、865.9 nm。和幼叶相比,成熟叶表面的粗糙度发生变化,但2个物种的变化趋势不同,这种变化可能与气孔的发育以及外界环境条件对叶片表面形态结构、蜡质含量和成分的影响不同有关。叶片表面存在大量的沟状、孔状峰谷区域和直径约为10 μm的凹陷,有利于PM₁₀的滞留。女贞和珊瑚树成熟叶气孔只分布在叶下表皮且下陷。这些特征均说明女贞和珊瑚树具有较强的滞尘能力和抵抗干旱、污染物胁迫的能力,作为绿篱植物对消减城市大气颗粒物污染和提高空气质量具有重要的意义。     

An in situ study of the nanomechanical properties of barnacle (Balanus amphitrite) cyprid cement using atomic force microscopy (AFM)

Abstract

Cyprids are the final planktonic stage in the larval dispersal of barnacles and are responsible for surface exploration and attachment to appropriate substrata. The nanomechanical properties of barnacle (Balanus amphitrite) cyprid permanent cement were studied in situ using atomic force microscopy (AFM). Force curves were recorded from the cement disc continually over the course of its curing and these were subsequently analysed using custom software. Results showed a narrowing of the pull-off force distribution with time, as well as a reduction in molecular stretch length over time. In addition, there was a strong correlation between maximum pull-off force and molecular stretch length for the cement, suggesting ‘curing’ of the adhesive; some force curves also contained a ‘fingerprint’ of modular protein unfolding. This study provides the first direct experimental evidence in support of a putative ‘tanning’ mechanism in barnacle cyprid cement.     

原子力显微技术在酶学研究中的应用

酶在生物体的生命活动中占有及其重要的地位,机体功能的和谐统一有赖于酶的作用。原子力显微技术(AFM)作为一门新发展起来的技术,为人们认识酶的结构与功能提供了又一新的窗口。AFM能够在生理条件下对生物样品进行三维成像,在分子水平上实时监测生理生化反应。AFM还能够在皮牛顿精度上测定分子间作用力。目前,AFM已用于单分子酶的化学性质及其作用原理的研究。本简述AFM在酶学中的应用情况。     

Atomic force microscopy (AFM) imaging suggests that stromal interaction molecule 1 (STIM1) binds to Orai1 with sixfold symmetry

Depletion of Ca²⁺ from the endoplasmic reticulum (ER) lumen triggers the opening of Ca²⁺ release-activated Ca²⁺ (CRAC) channels at the plasma membrane. CRAC channels are activated by stromal interaction molecule 1 (STIM1), an ER resident protein that senses Ca²⁺ store depletion and interacts with Orai1, the pore-forming subunit of the channel. The subunit stoichiometry of the CRAC channel is controversial. Here we provide evidence, using atomic force microscopy (AFM) imaging, that Orai1 assembles as a hexamer, and that STIM1 binds to Orai1 with sixfold symmetry. STIM1 associates with Orai1 in the form of monomers, dimers, and multimeric string-like structures that form links between the Orai1 hexamers. Our results provide new insights into the nature of the interactions between STIM1 and Orai1.     

Probing Interactions within the synaptic DNA-SfiI complex by AFM force spectroscopy

SfiI belongs to a family of restriction enzymes that function as tetramers, binding two recognition regions for the DNA cleavage reaction. The SfiI protein is an attractive and convenient model for studying synaptic complexes between DNA and proteins capable of site-specific binding. The enzymatic action of SfiI has been very well characterized. However, the properties of the complex before the cleavage reaction are not clear. We used single-molecule force spectroscopy to analyze the strength of interactions within the SfiI-DNA complex. In these experiments, the stability of the synaptic complex formed by the enzyme and two DNA duplexes was probed in a series of approach-retraction cycles. In order to do this, one duplex was tethered to the surface and the other was tethered to the probe. The complex was formed by the protein present in the solution. An alternative setup, in which the protein was anchored to the surface, allowed us to probe the stability of the complex formed with only one duplex in the approach-retraction experiments, with the duplex immobilized at the probe tip. Both types of complexes are characterized by similar rupture forces. The stability of the complex was determined by measuring the dependence of rupture forces on force loading rates (dynamic force spectroscopy) and the results suggest that the dissociation reaction of the SfiI-DNA complex has a single energy barrier along the dissociation path. Dynamic force spectroscopy was instrumental in revealing the role of the 5 bp spacer region within the palindromic recognition site on DNA-SfiI in the stability of the complex. The data show that, although the change of non-specific sequence does not alter the position of the activation barrier, it changes values of the off rates significantly.     

Quasi-linear viscoelastic properties of costal cartilage using atomic force microscopy

Costal cartilage (CC) is one of the load-bearing tissues of the rib cage. Literature on material characterisation of the CC is limited. Atomic force microscopy (AFM) has been extremely successful in characterising the elastic properties of soft biomaterials such as articular cartilage and hydrogels, which are often the material of choice for cartilage models. But AFM data on CC are absent in the literature. In this study, AFM indentations using spherical beaded tips were performed on human CC to isolate the mechanical properties. A novel method was developed for modelling the relaxation indentation experiments based on Fung's quasi-linear viscoelasticity and a continuous relaxation spectrum. This particular model has been popular for uniaxial compression test data analysis. Using the model, the mean Young's modulus of CC was found to be about 2.17, 4.11 and 5.49 MPa for three specimens. A large variation of modulus was observed over the tissue. Also, the modulus values decreased with distance from the costochondral junction.     

Fine mapping of inherent flexibility variation along DNA molecules. Validation by atomic force microscopy (AFM) in buffer

Curvature and flexibility are structural properties of central importance to genome function. However, due to the difficulties in finding suitable experimental conditions, methods for studying one without the interference of the other have proven to be difficult. We propose a new approach that provides a measure of inherent flexibility of DNA by taking advantage of two powerful techniques, X-ray crystallography and nuclear magnetic resonance. Both techniques are able to detect local curvature on DNA fragments but, while the first analyzes DNA in the solid state, the second works on DNA in solution. Comparison of the two data sets allowed us to calculate the relative contribution to flexibility of the three rotations and three translations, which relate successive base pair planes for the ten different dinucleotide steps. These values were then used to compute the variation of flexibility along a given nucleotide sequence. This allowed us to validate the method experimentally through comparisons with maps of local fluctuations in DNA molecule trajectory constructed from atomic force microscopy imaging in solution. We conclude that the six dinucleotide-step parameters defined here provide a powerful tool for the exploration of DNA structure and, consequently will make an important contribution to our understanding of DNA-sequence-dependent biological processes.     

Exploring the mechanical properties of single vimentin intermediate filaments by atomic force microscopy

Intermediate filaments (IFs), together with actin filaments and microtubules, compose the cytoskeleton. Among other functions, IFs impart mechanical stability to cells when exposed to mechanical stress and act as a support when the other cytoskeletal filaments cannot keep the structural integrity of the cells. Here we present a study on the bending properties of single vimentin IFs in which we used an atomic force microscopy (AFM) tip to elastically deform single filaments hanging over a porous membrane. We obtained a value for the bending modulus of non-stabilized IFs between 300 MPa and 400 MPa. Our results together with previous ones suggest that IFs present axial sliding between their constitutive building blocks and therefore have a bending modulus that depends on the filament length. Measurements of glutaraldehyde-stabilized filaments were also performed to reduce the axial sliding between subunits and therefore provide a lower limit estimate of the Young's modulus of the filaments. The results show an increment of two to three times in the bending modulus for the stabilized IFs with respect to the non-stabilized ones, suggesting that the Young's modulus of vimentin IFs should be around 900 MPa or higher.     

Observation of single-stranded DNA on mica and highly oriented pyrolytic graphite by atomic force microscopy

Atomic force microscopy was used to image single-stranded DNA (ssDNA) adsorbed on mica modified by Mg(2+), by 3-aminopropyltriethoxysilane or on modified highly oriented pyrolytic graphite (HOPG). ssDNA molecules on mica have compact structures with lumps, loops and super twisting, while on modified HOPG graphite ssDNA molecules adopt a conformation without secondary structures. We have shown that the immobilization of ssDNA under standard conditions on modified HOPG eliminates intramolecular base-pairing, thus this method could be important for studying certain processes involving ssDNA in more details.     

颗石藻颗石粒形态的原子力显微观测方法: 以赫氏艾密里藻为例

颗石藻(coccolithophore)作为一种模式生物, 在重建古海洋气候和环境以及预测未来全球气候变化中起着很重要的作用, 赫氏艾密里藻(Emiliania huxleyi)是颗石藻最为典型的代表种。钙质颗石粒(coccolith)是颗石藻形态分类的主要依据, 有着非常精细和复杂的结构, 在样品收集过程中很容易遭到破坏, 这是颗石藻鉴定中经常遇到的一个技术问题。国际上还没有统一的颗石藻定量采样和样品分析方法。本文采用原子力显微方法(atomic force microscopy, AFM)对赫氏艾密里藻的颗石粒形态进行了超显微观察研究, 获取不同扫描范围的高度图(height image)和形貌图(deflection image)以观测其形态结构, 并建立了针对颗石藻的原子力显微样品制备方法。通过离心与膜过滤两种方法收集赫氏艾密里藻, 比较后得出了一种简单、快速的适合于观测颗石藻在大气环境成像的样品处理、制备和图像采集方法: 3,000-4,000 rpm, 20℃离心5 min, 收集颗石藻, 去除有机杂质后取白色沉淀, 将沉淀物悬浮于0.05 M NH₄HCO₃溶液中, 悬浮液滴加于盖玻片表面, 20℃晾干后于样品台在AFM接触模式(contact mode)下原子级扫描, 扫描范围50 µm, 频率1 Hz, 可以得到优质的颗石粒形态图像, 有助于颗石藻的分类鉴别。该方法可用于室内不同环境梯度或参数下的颗石粒形态结构及颗石藻藻华的检测与研究。     

Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy

A novel method for the covalent attachment of erythrocytes to glass microscope coverslips that can be used to image intact cells and the cytoplasmic side of the cell membrane with either solid or liquid mode atomic force microscopy (AFM) is described. The strong binding of cells to the glass surface is achieved by the interaction of cell membrane carbohydrates to lectin, which is bound to N-5-azido-2-nitrobenzoyloxysuccinimide (ANBNOS)-coated coverslips (1). The effectiveness of this method is compared with the other commonly used methods of immobilizing intact erythrocytes on glass coverslips for AFM observations. Experimental conditions of AFM imaging of biologic tissue are discussed, and typical topographies of the extracellular and the cytoplasmic surfaces of the plasma membrane in the dry state and in the liquid state are presented. Comparison of the spectrin network of cell age-separated erythrocytes has demonstrated significant loss in the network order in older erythrocytes. The changes are quantitatively described using the pixel height histogram and window size grain analysis.     

Quantitative characterization of single‐cell adhesion properties by atomic force microscopy using protein‐functionalized microbeads

A method was developed to characterize the adhesion properties of single cells by using protein‐functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin‐functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM‐based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.     

Piezoelectric tuning fork probe for atomic force microscopy imaging and specific recognition force spectroscopy of an enzyme and its ligand

Piezoelectric quartz tuning fork has drawn the attention of many researchers for the development of new atomic force microscopy (AFM) self‐sensing probes. However, only few works have been done for soft biological materials imaging in air or aqueous conditions. The aim of this work was to demonstrate the efficiency of the AFM tuning fork probe to perform high‐resolution imaging of proteins and to study the specific interaction between a ligand and its receptor in aqueous media. Thus, a new kind of self‐sensing AFM sensor was introduced to realize imaging and biochemical specific recognition spectroscopy of glucose oxidase enzyme using a new chemical functionalization procedure of the metallic tips based on the electrochemical reduction of diazonium salt. This scanning probe as well as the functionalization strategy proved to be efficient respectively for the topography and force spectroscopy of soft biological materials in buffer conditions. Copyright © 2013 John Wiley & Sons, Ltd.