我们感谢陈懿范医生帮助的轴向光镊和他的拉伸数据，这份手稿的贡献。这项工作是由美国国家科学基金会授予的PHY-0957293，FOCUS授予PHY-0114336主办。

1。镊子设置<ol><li>从1064 nm的激光的光束被分成两个正交的偏振光束。一个用来操纵所述生物分子，而另一种是用于校准的目的（参见图2）。 </li><li>操纵光束的强度的控制由一个声光偏转器（AOD），而每束光的位置和焦点被独立地控制由光束转向反射镜和光学望远镜，分别。 </li><li>的光束，然后再结合另一偏振分束器和最后一组的伸缩光学稍微过量填充背面的显微镜物镜的孔径进一步空调。用于校准光束的50％的过满导致紧的光阱，而略小的因素的操纵光束，约20％，给出了一个浅的焦点，转换成一个较大的可行的区域中的线性电位。梁紧紧围绕一个PlanApo 60x/1.4的油浸显微镜obj的ective到样品池。 </li><li>这镊子设置结合构建家庭提供照明的明视野显微镜，从卤素灯聚焦到样品室由冷凝器。明场图像分离从激光光由分色镜和两个CCD相机成像，然后。一个CCD作为我们采集数据的主要手段是软件触发，得到精确的采样率，而其他CCD使用一个单一的卡住的参考微球的位置作为一个反馈控制系统补偿漂移在显微镜图像。 </li><li>将样品粗定位相对于通过XY载物台的目标，然后，可以精确地定位由一个嵌入三维压电阶段。 </li><li>的前向散射和发射的激光是由明场冷凝器收集，并集中到一个光检测器。过滤由此产生的信号通过一个抗混叠滤波器100千赫，扩增和cquired在200 kHz使用DAQ卡。 </li></ol> 2。校准视大小的珠轴向位置<ol><li>装入样品室9的直径为800纳米的聚苯乙烯微球的分散溶液稀释于磷酸盐缓冲盐水。让我们坐10至15分钟，然后轻轻地刷新缓冲区以清除多余的球。 </li><li>找到随机粘贴至盖玻片的微球，和调整的样品腔室的高度，直到大约1微米的微球是下方的焦点以产生散焦的图像。 </li><li>要测量的表观大小的图像，先找到中心的微球在LabVIEW中使用几何图案匹配功能。 </li><li>接着，生成的微球的径向强度的档案通过平均每个横截面的中心的约360°。的径向轮廓，这对应于在每个的明视野图像的白色环，可以配备一个二次函数，找到亮度峰值。此峰和中心之间的距离，可以使用作为衡量的视在尺寸的胎圈。 </li><li>使用校准piezostage，逐渐增加的微球的轴向位置，并在每个轴向位置与CCD摄像机获取的图像。 </li><li>重复的图像分析，对于每个连续的图像相关联的微球的表观大小与它的轴向位置（参照图3）。法求得的轴向分辨率为1.4 nm左右，7。 </li></ol> 3。映射的操纵光束的光学势<ol><li>首先通过共线对准的操纵光束校准光束。校准光束操纵束在后面光圈的目标是50毫瓦左右，而应该是弱得多，说10毫瓦。 </li><li>操纵光束关闭，限制一个免费的微球内的校准光束更严厉的陷阱。 </li><li>现在，打开歧管pulation束。由于操纵光束是远弱于校准光束的微球的轴向位置会稍有扰动。如已经描述的散焦的明视野图像，由此产生的轴向位置的变化，可以测量。 </li><li>关闭操作束微球仍被困在校准光束，调整的伸缩式镜头转向轴的校准光束的焦点。打开操纵束，测量后的微位移，并重复。 </li><li>的位移ΔX可以绘制对校准光束的焦点的轴向位置。对应的微球的最大位移的轴向位置确定的线性区域的光阱（参照图4）的中心。 </li><li>的最后步骤中获得的光作为轴向位置的函数的操作束力的刚性，需要仔细的测量校准光束。要获得此，周围的千分尺的表面上方移动的微球，被困由校准束，通过调节的伸缩式镜头，而操纵束关闭。 </li><li>通过测量与光检测器的透射光的强度，记录的微球的热的轴向运动。 </li><li>可安装到一个单一的指数衰减函数的信号的自相关性的波动，得到的时间常数，τZ </li><li>的液力动压摩擦系数ζ的微球可以被校正为微球接近的表面5,10。然后给出κZ =ζ/τZ（见补充材料A和B）的校准器的刚度。 </li><li>知道校准陷阱的刚度，允许一个先前测量的轴向位移转换成力ƒZ =κŽΔX整合氏s曲线操纵束（参照图4）的轴向的光学势产生的空间映射。 </li></ol> 4。拉伸的DNA样品<ol><li>安装室9含有表面拴系的DNA分子（&lt;1 kbp的），并同时观察明场图像，操纵光束的焦点放置略高于未拉伸的微球体通过调整望远镜。被捕获到的微球，然后调整位置的阶段。 </li><li>粗略地定位在微球中的XY平面上的光陷阱中心。产生一系列在LabView方波，和将它们发送到的AOD重复地打开和关闭上的激光束。小心地控制激光的强度和持续时间上的状态的样品腔室，以防止加热。通常情况下，使用约2 mW的激光功率（在后面光圈的客观测量），并传送脉冲的200毫秒（on）和500毫秒（关闭）。的振幅的平方瓦特大街发送到AOD约为0.5 V。 </li><li>关注微球的陷阱反复打开和关闭，注意，如果激光诱导任何优惠的微球的运动方向。虽然迭代调节微球体在X和Y方向的位置，通过控制piezostage，的微球的随机运动应该成为各向同性的在XY平面上，虽然明显的限制，当激光上。 </li><li>接着，在Z方向上对齐胎圈。再次脉冲激光束的开和关，而这个时间，同时测量实时的微球的轴向位移。中心的线性区域内的阶段，这是其中Z位移为最大的点。 </li><li>在Z轴方向进行精心调整后，当务之急是要检查的陷阱仍然集中在XY平面上。如果已经改变，XY对准XY和Z对准的微球必须被重复，直到正确的中心沿三个轴。 </li><li>对于拉伸的DNA，坡道的激光强度的AOD发送的电压信号，从0 V至0.5 V，在步骤0.025 V.在每个步骤中的，记录400在100帧的帧和平均他们获得的轴向位移。 </li><li>力拓曲线可以绘制适合改良的WLC模型11（见补充材料B）。 </li></ol> 5。代表性的成果： 力拓曲线的两个DNA序列：一个1298 bp和247 bp的序列，后者在最短的时间序列已经能够伸展重复性的。对于短的DNA片段，传统的蠕虫状链（WLC）模型并不能完全解释力拓的关系，因为在这些尺度必须考虑的有限尺寸效应和零力拓所产生的边界约束。力拓测量，因此，必须要适合使用修改后的WLC模型，其中有一个有效的已经持效期长和零力拓拟合参数的补充材料，进一步说明。对于大的轮廓长度的双链DNA，有效的持久性的长度是简单的名义持续长度（约50 nm）和的零力拓可以忽略不计。然而，作为轮廓的长度变短有效的持久性的长度降低远低于50nm，并且该DNA，甚至零力下，示出了显着的延伸。 的数据和相应的配合力拓曲线示于图中。 5为两个序列。从改性WLC适合，我们已经确定了有效的持久性长度为35 nm的1298 bp的DNA为247 bp的DNA和25 nm。为了说明的目的，我们将呈现的每个序列的力扩展曲线的单一的措施。在实践中，人们会重复测量多次，得到的平均结果与标准误差一起。还必须指出的临屋区t后获得每条曲线，它是必须确保的微球保持捕集并正确定位的线性区域内，否则必须重新调整的微球，如先前描述的协议。 <img alt="图1" src="/files/ftp_upload/3405/3405fig1.jpg" /> 图1（左） 的轴向光镊原理传统的光学镊子陷阱附近的重点。珠，然后横向盖玻片移动，施加张力。 （右）在轴向光镊，微球被困住远离焦点，在光学势的线性区域。在此配置中，该聚合物被保持在恒定的张力下的范围内的扩展。此外，增加的激光强度，在轴向方向上移动的微球。 <img alt="图2" src="/files/ftp_upload/3405/3405fig2.jpg" /> 图2示意图轴向光镊激光灯（1064纳米的Nd：YVO 4）被分成两束，通过通过一个偏振分束器（PBS）。的操纵光束，通过一个声光偏转器（AOD），使用伸缩式镜头校准光束都可以独立调节。的两束，然后重新组合使用另一种的PBS，并聚焦在样品由高NA物镜。同时，明视野照明，用于跟踪的微球，通过样品，是红外（IR），过滤，然后由两个CCD相机，其中之一作为一个反馈控制系统的一部分，而其他行为进行测量成像。 <img alt="图3" src="/files/ftp_upload/3405/3405fig3.jpg" /> 图3轴向定位校准要校准的轴向位置，我们获得了离焦图像珠坚持腔室盖玻片，在不同阶段的轴向位置。的大小的微phere其中心和周围形成的微球的图像的亮环的峰强度的位置之间的距离由下式给出。插图呈现的径向光强分布，其中给出的胎圈的大小。 <img alt="图4" src="/files/ftp_upload/3405/3405fig4.jpg" /> 图4映射原理的光学势。要映射出光操纵束力，在微被困在一个更强大的校准光束，它引起的轴向位移测量。的线性区域的中心，其中该位移是一个最大的位置。可以集成的光学势的光学力与轴向定位曲线。 <img alt="图5" src="/files/ftp_upload/3405/3405fig5.jpg" /> 图5力拓曲线。数据显示为一个随机的1298基点和247基点（插图）节段吨双链DNA拉伸轴光学镊子。适合修改后的WLC模型式的数据点。 3（实线），并取得了有效的持久性为34 nm和25 nm的长度分别为1298bp和247bp。

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没有利益冲突的声明。

传统的光学镊子依赖于模拟或电脑控制的反馈上的折光性对象施加一个恒定的力。这些有源反馈系统有困难的其中的突然变化在延长的试样发生的条件下进行，例如，从结合的蛋白质的DNA或一个分子马达沿长丝的快速步进。最近已开发用于施加恒定力的各种无源方法。涉及一种这样的方法，用来解决碱基对组成的RNA聚合酶的步进分辨率的光学势高斯激光束12的线性区域内工作。我们已经适...

<table border="1"><tbody><tr><th>试剂/设备</th><th>公司</th><th>目录号</th><th>评论</th></tr><tr><td> Nd：YVO4激光器</td><td>光谱物理</td><td> T40-Z-106C </td><td></td></tr><tr><td>声光 导流板</td><td> IntraAction </td><td> DTD-274HA6 </td><td></td></tr><tr><td>显微物镜</td><td>奥林巴斯</td><td> PlanApo </td><td> 60X，NA 1.4 </td></tr><tr><td>压电阶段</td><td>疯狂的城市实验室</td><td>纳米LP100 </td><td> XYZ阶段</td></tr><tr><td> CCD相机</td><td> PixeLink </td><td> PL-A741 </td><td></td></tr><tr><td>光电探测器</td><td>光电 技术</td><td> ET-3020 </td><td></td></tr><tr><td>聚苯乙烯珠</td><td> Spherotech </td><td> SVP-08-10 </td><td> 800纳米，链霉亲和素 涂</td></tr><tr><td>抗地高辛</td><td>罗氏公司</TD&gt; <td> 11333089001 </td><td>从羊</td></tr><tr><td>引物</td><td> MWG操纵子</td><td>自定义的寡核苷酸</td><td>一个引物：生物素 其他：地高辛</td></tr><tr><td> PCR试剂</td><td>新英格兰 生物实验室</td><td> TAQ聚合酶， 的dNTPs </td><td></td></tr><tr><td>盖玻片</td><td> Fisher Scientific则</td><td></td><td></td></tr><tr><td>其他化学品 缓冲</td><td> Fisher Scientific则</td><td></td><td></td></tr></tbody></table>补充材料 A.液力摩擦系数用于确定的微球的水动力的摩擦系数的表面附近，1可以使用下面的膨胀5,10： <img src="/files/ftp_upload/3405/3405eq1.jpg" alt="式（1）" />以下简写Ĥ作为介绍： <img src="/files/ftp_upload/3405/3405eq2.jpg" alt="公式2" />的摩擦系数的定义中的流体的粘度η的微球的半径，与微球的中心位于距离η的表面之上。的总和收敛合理扩大到约10条款。 B.刚度标定的轴向位置的影响陷阱刚度涉及校准校准的精度，从而增加与距表面的距离的增加，和实际的轴向位置的陷阱实验使用之间的权衡。在一般情况下，陷阱被校准，从高于实际的实验条件下的表面，这是在约800-1000纳米。 C.改进的蠕虫状链（WLC）模型力拓曲线可以适应修改后的WLC米ODEL占体积排阻效应在零光力如下： <img src="/files/ftp_upload/3405/3405eq3.jpg" alt="式（3）" /> 其中 ，F opt是光学的力中，x o是一个配合的参数的零力延伸中，x opt是下的延伸力，l是长度的DNA的轮廓，和升* p是第二个适合的“有效的参数为“持效期长。f WLC是通常WLC模型11 <img src="/files/ftp_upload/3405/3405eq4.jpg" alt="式（4）" /> 其中ε是相对的DNA延伸。

stretching short sequences of dna with constant force axial optical tweezers

拉伸DNA的轮廓长度小于一个碱基的单分子技术是充满了实验的困难。然而，许多有趣的生物事件，如组蛋白结合的DNA 1,2和蛋白介导的循环，出现该长度的规模。近年来，DNA的机械性能已被证明是在基本的细胞过程中起到显着的作用类似的包装成紧凑的核小体的DNA和染色质的纤维3,4。显然，这是重要，了解短的DNA片段的力学性能。在本文中，我们提供了一个实用指南如何运用眼线改变单分子光学技术，我们已经开发研究的力学行为与轮廓长度短到几百个碱基对的DNA。 伸展短的DNA片段的主要障碍是，传统的光学镊子的设计，一般使用武力中的directioŇ横向的阶段5,6，（参见图1）。在这种几何形状，胎圈和盖玻片之间的角度，其中DNA是拴，变得很陡的亚微米长度的DNA。必须现在可以占的轴向位置，这可以是一个挑战，并且，由于扩展的微球拖动接近盖玻片，空间位阻效应增强。此外，作为微球的不对称的结果，横向扩展，会产生不同水平的转矩由于由于在延长的方向上的反作用力的变化的光陷阱内的微球的旋转。 拉伸亚微米DNA的替代方法，对自己的独特的障碍。比如，双束光阱是有限的拉伸周围的波长，在该DNA的两个陷阱的点之间的干涉效应和从光散射之间的微球体开始构成了显着的问题，。更换一个陷阱用微量最有可​​能遭受类似的挑战。虽然人们可以直接使用的轴向电位伸展DNA，有源反馈方案将需要施加一个恒定的力的带宽，这将是相当有限的，尤其是在低力量。 我们规避这些基本问题直接拉的DNA，从盖玻片上使用恒定的力的轴向光镊7,8。这是通过捕集的胎圈在光学势的线性区域，其中的光学力是恒定的强度可以通过调整激光功率调谐。俘获的线性区域内还用作所有的光学近350nm的在轴向方向上延伸的DNA上的力夹紧。我们同时补偿通过微调整的位置的阶段，这样的热性能和机械漂移盖玻片粘贴至一个参考微球保持在相同的位置和焦点，一个llowing一个几乎是无限的观察期。

Watch this Scientific Journal Video about Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers at JoVE.com

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

我们说明了使用一个恒定的力的轴向光镊探索短的DNA分子的力学性能。轴向拉伸DNA，我们尽量减少在传统的横向操作所产生的立体障碍和文物，使我们能够研究DNA分子的短〜100 nm之间。

伸展的DNA短序列恒力轴向光镊

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

stretching-short-sequences-dna-with-constant-force-axial-optical

Research

JoVE Journal

Bioengineering

13.0K 次观看.  University of Michigan. 我们说明了使用一个恒定的力的轴向光镊探索短的DNA分子的力学性能。轴向拉伸DNA，我们尽量减少在传统的横向操作所产生的立体障碍和文物，使我们能够研究DNA分子的短〜100 nm之间。

视频: Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

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

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

警队复用单分子蛋白水解测量磁性镊子

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

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 ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

生物传感器检测抗生素耐药金黄色葡萄球菌细菌

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

定量的光学显微镜：测量细胞生物物理特性与标准光学显微镜

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

在硅橡胶膜拉伸微图案细胞

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

磁性镊子的扭曲和扭矩的测量

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

单个RNA分子的光镊纳米操作

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

使用基于曲线的转换工具量化纤维胶原蛋白组织

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...