这项研究是，美国能源部生物和环境研究办公室，并授予来自弗吉尼亚州的联邦卫生研究委员会的资金赞助。美国能源部橡树岭国家实验室管理有限责任公司，UT斯达康，巴特尔，根据合同号DE - AC05 - 00OR22725。

1。云母的准备： <ol><li>削减云母的大小要适合AFM显微镜（约22 × 30毫米）的剪刀（电子显微镜学）。 </li><li>顺劈斩双方云母，一般用胶带去除的外层，直到顺利不间断层保持。 </li></ol> 2。明胶溶液的制备： <ol><li>实验室瓶100毫升的蒸馏水。 </li><li>一瓶在微波加热，直到水开始沸腾。 （如果不可用微波炉，水可以在一个热点板块的烧杯加热。） </li><li>称取0.5克，明胶（Sigma公司的G - 6144 G - 2625或G - 2500），加入热蒸馏水。 </li><li>轻轻摇动瓶子直到明胶完全溶解。 </li><li>明胶溶液冷却到60-70 ° C。 </li><li>削减云母可斜射到小烧杯中（20毫升）倒入约15毫升。 </li></ol> 3。云母涂层： <ol><li>云母广场淹没到温暖的明胶溶液，并迅速撤出，云母涂层（图1a）。 </li><li>涂装后，立即支持云母，边缘，在纸巾上，周围空气中的干（图1b）。明胶包覆云母可用于至少2周。多余的明胶溶液可冷藏保存，使用约一个月只需再加热原液之间60-70 ° C。必须小心，以确保明胶期间未再加热煮沸。 </li></ol> 4。安装明胶包覆云母的细菌： <ol><li>佩莱1毫升细菌培养在800至4​​500 RCF（0.5 - 1.0在600 nm的OD）。根据上的细菌，使用最低的区域合作框架，将沉淀的细胞〜5分钟。 </li><li>在相同体积的过滤去离子水或缓冲液洗涤沉淀。 </li><li>再通过离心收集细胞。 </li><li>及时nanopure去离子水或缓冲液500μL重悬沉淀。 （菌悬液应明显混浊才能有足够的浓度细胞的原子力显微镜成像。） </li><li>应用细胞悬液（10-20μL）明胶包覆云母和表面上的传播，使用吸头（图1c）。应采取不接触枪头明胶的表面，而申请或传播细菌的解决方案。 </li><li>允许休息10分钟，并与流的水或缓冲液（图1 CD）冲洗表面。整个过程中，照顾，以确保该样本是不允许干的。 </li></ol> 5。代表性的成果： 图2显示了已安装在明胶包云母和原子力显微镜成像的细菌细胞的例子图像。在我们的实验室，样品通常使用一个PicoPlus原子力显微镜（安捷伦科技，寺，AZ），配备一个100微米的扫描头成像。该仪器操作在0.5-1.0行/秒的扫描速度在128-512像素，每行扫描通常使用的悬臂Veeco的氮化硅探针与标称弹簧常数的范围从0.01至0.1 NN /纳米（MLCT AUHW，Veeco公司，圣巴巴拉分校，加利福尼亚州）。 <img src="/files/ftp_upload/2880/2880fig1.jpg" alt="图1"/> 图1，在温度60-70℃放置在20毫升的烧杯中明胶。使用镊子新鲜剥离的云母广场完全沉浸到明胶（一）撤销，而直立靠在一个微型离心机机架（二）一纸毛巾放在。细菌样品液滴放置在X和Y方向（C）吸管尖云母和蔓延。使样品在云母表面孵育至少10分钟后，冲洗样品在蒸馏水流。如果固定工作，你将能够看到一个不透明的点上的云母，在右侧面板（D）所示。如果固定没有工作，你会得到明确的云母片，在左侧面板。云母鳞片白斑液滴的细菌浓度，明胶处理的云母。 <img src="/files/ftp_upload/2880/2880fig2.jpg" alt="图2"/> 图2 E 的 AFM图像。 大肠杆菌 。面板（A），细胞安装在明胶包云母和0.005 M PBS成像。为了便于比较，明胶包覆云母和安装在空气中成像的细菌细胞的图像显示面板（B）。虽然隔的液体，更高的分辨率菌毛（插图面板（二））表示，在采集到的图像中清楚可见，可在空气中获得。

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+axons.">Mechanical properties of axons.</a> Phys Rev Lett. 99, 018301-018301 (2007).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+in+response+to+experimentally+applied+mechanical+tension.">Axonal growth in response to experimentally applied mechanical tension.</a> Dev Biol. 102, 379-389 (1984).</li><li>Chetta, J., Kye, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+dynamics+in+response+to+tensile+loading+of+mammalian+axons.">Cytoskeletal dynamics in response to tensile loading of mammalian axons.</a> Cytoskeleton (Hoboken). 67, 650-665 (2010).</li><li>Dennerll, T. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension+as+a+regulator+and+integrator+of+axonal+growth.">Tension as a regulator and integrator of axonal growth.</a> Cell Motil Cytoskeleton. 17, 6-10 (1990).</li><li>Heidemann, S. R., Buxbaum, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+as+a+regulator+of+axonal+development.">Mechanical tension as a regulator of axonal development.</a> Neurotoxicology. 15, 95-107 (1994).</li><li>Heidemann, S. R., Lamoureux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomechanics+of+axonal+development.">Cytomechanics of axonal development.</a> Cell Biochem Biophys. 27, 135-155 (1995).</li><li>Iwata, A., Browne, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+survival+and+outgrowth+of+mechanically+engineered+nervous+tissue+constructs+implanted+into+spinal+cord+lesions.">Long-term survival and outgrowth of mechanically engineered nervous tissue constructs implanted into spinal cord lesions.</a> Tissue Eng. 12, 101-110 (2006).</li><li>Lamoureux, P., Heidemann, S. 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B., Franze, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+individual+glial+cells+and+neurons+in+the+CNS.">Viscoelastic properties of individual glial cells and neurons in the CNS.</a> Proc Natl Acad Sci U S A. 103, 17759-17764 (2006).</li><li>O'Toole, M., Lamoureux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physical+model+of+axonal+elongation:+force,+viscosity,+and+adhesions+govern+the+mode+of+outgrowth.">A physical model of axonal elongation: force, viscosity, and adhesions govern the mode of outgrowth.</a> Biophys J. 94, 2610-2620 (2008).</li><li>Pfister, B. J., Bonislawski, D. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch+growth+of+integrated+axon+tracts:+extremes+and+exploitations.">Stretch growth of integrated axon tracts: extremes and exploitations.</a> Prog Neurobiol. 89, 231-239 (2009).</li><li>Smith, D. H., Wolf, J. 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没有利益冲突的声明。

许多因素可能会影响微生物细胞固定的原子力显微镜和成像。明胶，用于涂料，云母是重要的。商业明胶是孤立的，从脊椎动物，包括鱼，牛，猪的数量。的起源和处理方法，确定为细菌固定化明胶的适用性。许多来源和类型的明胶固定细菌的有效性进行评估[1]。两个最有效的明胶被发现SIGMA的G - 6144和G - 2625。从猪（猪），这些明胶被认为是低布鲁姆和中等布鲁姆分别。明胶处理过的基材和特定?...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>明胶</td><td> Sigma公司，圣路易斯，密苏里州</td><td> G6144，G2625或G2500 </td></tr><tr><td> PicoPlus原子力显微镜</td><td>安捷伦科技公司，坦佩，AZ </td><td></td></tr><tr><td>原子力显微镜的悬臂</td><td> Veeco公司，圣巴巴拉，CA </td><td> MLCT - AUHW </td></tr></tbody></table>

bacterial immobilization for imaging by atomic force microscopy

AFM是一个高分辨率（纳米尺度）的成像工具，机械探头表面。它具有图像细胞和生物分子的能力，在液体环境中，无需化学处理的样品。为了实现这一目标，必须充分坚持样品安装表面，以防止去除扫描原子力显微镜悬臂的针尖施加的力量。在许多情况下，成功的成像取决于样品固定在安装表面。理想情况下，固定应微创样品等的代谢过程和功能属性不受损害。猪（猪）明胶涂层新鲜剥离的云母表面带负电荷的细菌可以固定在表面上，并在液体中原子力显微镜成像。明胶包覆云母上的细菌细胞固定化是最有可能是由于带负电荷的细菌和带正电荷的明胶之间的静电相互作用。有几个因素可以干扰细菌固定化，包括在其中的细菌悬浮液，明胶包覆云母，菌株和成像在其中的细菌培养基的表面特性的细菌培养时间化学成分。总体而言，使用明胶包覆云母成像微生物细胞被普遍适用。

Watch this Scientific Journal Video about Bacterial Immobilization for Imaging by Atomic Force Microscopy at JoVE.com

Bacterial Immobilization for Imaging by Atomic Force Microscopy

现场革兰氏阴性和革兰氏阳性菌，可固定在明胶包覆云母和利用原子力显微镜（AFM）在液体中成像。

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

bacterial-immobilization-for-imaging-by-atomic-force-microscopy

Research

JoVE Journal

Bioengineering

17.2K 次观看.  Oak Ridge National Laboratory. 现场革兰氏阴性和革兰氏阳性菌，可固定在明胶包覆云母和利用原子力显微镜（AFM）在液体中成像。

视频: Bacterial Immobilization for Imaging by Atomic Force Microscopy

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

重组DNA和蛋白质复合物，用原子力显微镜的可视化

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

科研

生物工程

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

量化的内皮细胞糖复合物的力学性能，用原子力显微镜

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

新型原子力显微镜基于生物淘选的形态特殊试剂对TDP-43变型肌萎缩侧索硬化症隔离

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

多显微镜下的血管生成术在基因修饰的 3 d-PLGA/nHAp 支架上进行颅骨临界骨缺损修复

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

牵引力显微镜研究B淋巴细胞激活

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

原子力显微镜在早期骨关节炎检测中的应用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...