这项工作是由美国国立卫生研究院NIDCD Y1-DC-8002-01的支持。根据合同DE-AC52-07NA27344美国能源署的主持下进行了劳伦斯利弗莫尔国家实验室这项工作。

1。探头大会补强本节中的协议的描述制造的硅加劲肋，并且薄膜的聚合物探针的装配到刚性元件， 图1示出了沿与提出的加强件的典型聚合物的神经探针。的加强筋设计的细节示于图2。这种设计的新颖特征是在浅“芯吸”通道沿其长度是用来在组装过程中分配液体粘合剂运行。加强板的较宽部分为装配和外科插入时处理的标签。该选项卡上的水库连接到的通道。该组件是由硅使用标准微制造工艺制造。 <ol><li>硅加强筋用​​芯吸通道是从一个硅 - 绝缘体（SOI）晶片上的器件层的厚度等于所述加强件的所需厚度（制造<strong&gt;的图3A）。一个合理的范围内加强筋厚度为20-100微米。建议加强筋的宽度比探针的宽度，这样有助于防止从键合界面的粘接剂溢出到该探针的顶端20-30微米以下。第一毛细作用通道进行干蚀刻使用标准的Bosch工艺（ 图3B）。接着，将加强件的几何形状是由一个较长的蚀刻掩埋氧化物层（ 图3C）上，停止所定义。最后，该加强件被释放通过在49％的氢氟酸（ 图3D）湿蚀刻掩埋氧化物层。后彻底冲洗加强筋，浸泡在去离子水中15分钟。 </li><li>把聚乙二醇的分子量为10,000克/摩尔的沉淀（PEG）进入贮存器（ 图4）。加热所述加劲肋至65℃，使聚乙二醇熔化，并通过毛细管作用灯芯进入通道。然后冷却至室温凝固。 </l我&gt; <li> 图5示出了倒装焊接机的示意性设置。将加强件上的倒装焊接机的基台倒置，然后拿起加强筋与工具头。把探头上的基台倒置。使用覆晶焊接机，对准加强筋和探头，然后放下加强筋，并将其放置在探头上。 </li><li>倒装焊接机的基台应该有施加热到所述衬底的加热元件。将所述加强板后，再次加热该组件至65℃。等待一分钟，对PEG重熔，并开始填写探头和加强筋之间的接口。冷却至凝固。 </li><li>打开装配过来，从顶部检查。根据需要，以允许所述PEG以完全填充所述探针和所述加强件之间的界面上重新加热。这可以在视觉上进行评估，因为探头是透明的。由于组件坐在加热器顶部（探针）朝上，手动放置1-3Ë固体PEG与Xtra的丸粒上的标签，使它们熔化过的探针，可提供在该区域的其他加强件（ 图6）。最后，请允许组件冷却，以使PEG凝固。在这一点上，该组件已准备好手术插入。 </li></ol> 2。插拔<ol><li>安装探针刚性组件到一个显微操作器通过粘附加强板的背面的显微操作臂在选项卡区域， 如图7A所示 。这可以用双面胶或水泥做的，但要注意不要接触探头用胶粘剂。探头的连接器端暂时固定在用一小片粘合剂腻子，使得它可以容易地用小的力去除的微操作。 </li><li>定位探针组件对目标以及与所需的插入速度插入探头。为0.13-0.5毫米/秒的速度插入发展这个协议的时候被使用。&lt;/ LI&gt; <li>立即从显微操纵器轻轻探头的连接器端取出并放于附近的表面，如第二操纵臂（ 图7B）。这是一定要做的PEG开始溶解，以避免取代前探。 </li><li>等待一段时间以PEG溶解。此量的时间将取决于PEG的分子量和探针与加强板之间的接触面积。例如，为10,000克/摩尔，微电极探针约6mm和匹配的加强件是306μm宽的PEG分子量，15分钟已发现的时间足够量。该协议的第3提出了一种方法来测试所要求的溶解时间。在此期间，应用磷酸盐缓冲盐水（PBS）用滴管周围的标签和插入点以溶解任何PEG即上面的目标（ 图7C）。 </li><li>使用机动微定位器，通过应用的位移开始提取的加强筋为100μm以5mm /秒的速度。这最初的快速运动有助于克服任何静摩擦和减少探针位移。然后，完成加强筋提取在大约0.1mm /秒（ 图7D）以较慢的速度。 </li><li>在实际手术的情况下，继续正常的程序申请凝胶，硅胶，和/或牙科丙烯酸在插入位点，以确保和保护探头，这表现在21。 </li></ol> 3。琼脂糖凝胶测试本节中的协议描述了一种设置和程序来检查加强筋中，接近本体的机械性能，pH值，和脑组织17,22的盐度为0.6％的琼脂糖凝胶提取。由于凝胶几乎是透明的，通过短距离，加强筋的分离和探针位移可以观察到。 <ol><li>制备在磷酸盐缓冲盐水（PBS）的0.6％琼脂糖溶液。在混合的元素vated温度以完全溶解琼脂糖粉末。将溶液倒入一个浅压克力盒;凝胶应该是3/4- 1深。允许对凝胶设置在室温一小时。 </li><li>确保硬化的凝胶是饱和的PBS，使其不会变干，然后将凝胶加热到37°C。 </li><li>设置微操作，框琼脂糖凝胶中，并且显微摄像系统， 如图8。 </li><li>插入玻璃参考基准成凝胶的盒由凝胶和盒子的侧面（ 图8）之间滑动。用牙科挑广场上的参考基准的功能，以视数码显微镜领域。 </li><li>安装探头组件的显微操纵器，如步骤2.1中所述。 </li><li>定位探针组件在凝胶约1mm后面的参考基准。 </li><li>将探头插入凝胶中，在使用相机来指导它在视场中所需的深度。&lt;/ LI&gt; <li>紧随探头的连接器端移动到休息在附近的表面。 </li><li>进行必要的调整摄像机图像聚焦探头（参考基准特征可能会稍微失焦）。取探头位置的快照。 </li><li>允许PEG溶解（此时可能会发生变化，而事实上可能是一个参数进行测试）。应用PBS附近的标签，以溶解聚乙二醇是凝胶上面。 </li><li>如果需要启动视频拍摄，并且按照步骤2.5中所述的提取开始加劲肋。解压缩完成后，把探头位置的最终快照。 </li><li>使用图像处理工具图像之前和之后加强筋抽出来比较。使用的参考基准是可见的，以登记该字段的功能（对齐）的图像。校准的基础上的探针上已知的特征的尺寸的图像的尺度。测量探头位移的距离。 </li></ol>

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该作者有没有竞争的财务权益。

这里描述的方法提供了一个良好的控制过程附加薄膜聚合物探针来分离加强筋与biodissolvable粘合剂。还提出，建议外科手术，以实现这些可移动的加强筋和技术验证过程在体外对于一个给定探针加强筋的配置。由于加强件可以由任意刚性的，该方法可以方便地插入相对长的探针（&gt; 3mm）的中。因此，该插入方法有望成为一个有利的技术在脑深部刺激（DBS），脊髓刺激，和外周神经接口的应...

微电极阵列在神经科学的一个重要工具，以及新兴的临床应用，如假肢。特别是，插入的微电极探针通过与细胞在大脑，脊髓和周围神经亲密接触使刺激神经元的活动和记录。对于植入式神经探针的一个主要挑战是稳定的刺激和记录功能，延年益寿。微电极探针和神经组织之间的相互作用的建模和实验研究表明，1机制降解是微撕裂神经组织，由于在探头和组织1-3之间轻微的相对运动。一种解决方案是制造匹配更紧密神经组织，以减少相对微动的体积压缩刚度特性灵活的探针。因此，生物相容的薄膜的聚合物，例如聚酰亚胺和聚对二甲苯已被采用为有利的基板为微电子处接上的电流探头4-8。 柔性探头的权衡是，他们是难以插入到神经组织。研究人员已经采取了各种方法，以便于插入的柔性探头，同时保留所希望的机械性能。一类设计的修改聚合物探针的几何形状，同时遵守其他地区，以增加刚度在某些部分或轴。这已经完成通过将肋或其它材料9,10层。另一种方法中集成了一个3-D信道到聚合物探针设计填充有可生物降解的材料11。该探针可以被临时硬化，并插入该材料在通道中溶解并排出，之后。然而，方法如上述那永久修改最终植入的装置的几何形状可能会损害一些的柔性探针的理想特征。 一种方法，做Ñ加时改变最终探针的几何形状是封装在聚合物装置与可生物降解的材料，以暂时硬化装置12-14。然而，典型的可生物降解材料具有的杨氏模量幅度比硅小的订单，并作相应需要更大的厚度，以达到相同的刚度。充分涂覆探针可产生更圆的或钝头，使得插入更加困难。此外，由于可分解涂层暴露，有他们的危险接触后立即溶解，甚至近在咫尺，与组织。 另一类方法采用新型探头的基板材料，以减少刚度被植入组织后。这样的材料包括形状记忆聚合物15和一个机械适应性纳米复合材料16。这些材料能够在弹性模量显著插入后下降，并可能导致探针更紧密MATC神经组织h的机械性能。然而，刚性的可实现的范围仍然是有限的，因此它们可能不能够提供非常高的刚度等于硅或钨丝。因此，在柔性探针是很长的（ 例如 &gt; 3毫米），或具有非常低的刚度的情况下，可能仍然需要的临时固定更刚性的加强筋的方法。 报道又一个有前途的方法是涂层具有永久自组装单层（SAM）来定制班车和灵活的探头17之间的表面相互作用的加强班车。在干燥时，探针附着于涂穿梭静电。插入后，水将迁移到该亲水表面，从航天飞机分离探针，使得往复可以被提取。穿梭萃取减小探针位移被证明（85微米）。然而，仅静电相互作用保持探头吨他穿梭，有探头打滑相对于航天飞机之前和期间插入一定的风险。 我们已经开发了在该柔性探头连接到与该牢固地保持在插入过程中探针的临时biodissolvable粘合剂材料的加强件的方法。所用的探针是聚酰亚胺，其具有2-4京帕数量级的弹性模量进行。加强件是由硅制成，具有〜为200GPa的弹性模量。在连接时，硅的刚性支配，便于插入。一旦插入到组织中，粘合剂材料溶解和加强件被提取到探针恢复到其初始的灵活性。我们选择了聚乙二醇（PEG）作为biodissolvable粘合材料。 PEG已被用于在植入的应用，如神经探针，组织工程和药物递送11,18,19。一些证据表明，PEG可以减轻神经炎症反应在脑组织18,20。相对于其他可能的材料，包括蔗糖，聚乳酸 - 共 - 乙醇酸（PLGA），和聚乙烯醇（PVA），聚乙二醇具有的溶解时间在生物流体，它是一个合适的规模对许多植入手术（数量级几十分钟，这取决于分子量）。此外，它是固体在室温和液体温度范围为50-65℃。这个属性使得它特别适合于我们的精密装配过程。此外，在17个类似的SAM所描述的，溶解PEG是亲水性的，有利于提取的加强筋。这种有利的做法是由一种新型的加强筋设计和有条不紊的装配工艺，确保均匀的粘合剂覆盖面和准确的和可重复的对齐方式启用。除了 ​​装配过程中，我们提出了在手术过程中实现可移动的加强件的方法，以及在体外程序来评价提取STI的ffener。 这里介绍的协议假定用户具有柔性聚合物微电极探针。关于这个探头的加强筋与组件的制造到加强筋的协议的一部分，假设获得的微细加工设备中常见的工具。有关插入和提取的协议可能会在神经科学为导向的实验室进行。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
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			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Polyethylene glycol, 10,000 g/mol</td>
			<td>Sigma Aldrich</td>
			<td>309028</td>
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			<td>Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9539</td>
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			<td>Flexible Sub-micron Die Bonder</td>
			<td>Finetech</td>
			<td>Fineplacer lambda</td>
			<td></td>
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			<td>Micromanipulator</td>
			<td>KOPF</td>
			<td>1760-61</td>
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			<td>Digital Microscope</td>
			<td>Hirox</td>
			<td>KH-7700</td>
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			<td>Dual Illumination Revolver Zoom Lens</td>
			<td>Hirox</td>
			<td>MXG-2500REZ</td>
			<td></td>
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			<td>Precision Motorized Actuator</td>
			<td>Newport</td>
			<td>LTA-HS</td>
			<td>w/ CONEX-CC controller</td>
		 </tr>
		</tbody>
 </table>

insertion of flexible neural probes using rigid stiffeners attached with biodissolvable adhesive

预期微电极阵列所做生物相容性薄膜聚合物的神经接口设备已经扩展功能寿命，因为柔性材料可以减少因微动不良组织反应。然而，他们的灵活性，防止它们被准确地插入到神经组织。本文介绍的方法，以一种灵活的微电极探针临时性地连接到刚性加强板使用biodissolvable聚乙二醇（PEG），以促进所述探针的精确，手术插入。一个独特的加强筋的设计允许对PEG粘合剂的均匀分布沿着探针的长度。倒装芯片接合，在微电子封装中使用的常用工具，能够进行精确的和可重复的对准和连接的探针与加强筋。探针和加强件被手术植入在一起，然后将PEG使其溶解，使得加强筋可以被提取离开探头到位。最后， 在体外试验方法用于评价加强筋提取脑组织的琼脂糖凝胶模型。这种方法植入已被证明更长的柔性探头特别有利的（&gt; 3毫米）。它还提供了植入双面柔性探头一个可行的方法。迄今为止，该技术已被用来获得从大鼠皮层各种体内的记录数据。

这种插入技术被用于与LLNL的薄膜聚酰亚胺探头，它已通过ISO 10993生物相容性标准，并适用于慢性植入相结合。一个典型的薄膜聚酰亚胺探针沿着与硅刚性元件是大约10毫米长的狭窄区域， 如图1所示。此加强筋具有沿其长度延伸的1毛细通道， 如图2，图3示出了用于创建该加强筋出硅的mimcrofabrication过程， 图4示出固体的PEG的沉淀，它被放置成片的油藏，通过倒...

Watch this Scientific Journal Video about Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive at JoVE.com

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

的柔性神经微探针插入是通过将探针以刚性加强板与聚乙二醇（PEG）使能。独特的装配工艺确保了统一的和可重复的附件。插入到组织中后，所述PEG溶解和加强件被提取。 体外试验方法评估在琼脂糖凝胶的技术。

柔性神经微探针的使用附加与Biodissolvable胶粘剂刚性加劲肋插入

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

insertion-flexible-neural-probes-using-rigid-stiffeners-attached-with

Research

JoVE Journal

Bioengineering

14.6K 次观看.  Lawrence Livermore National Laboratory. 的柔性神经微探针插入是通过将探针以刚性加强板与聚乙二醇（PEG）使能。独特的装配工艺确保了统一的和可重复的附件。插入到组织中后，所述PEG溶解和加强件被提取。 体外试验方法评估在琼脂糖凝胶的技术。 

视频: Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

将生物探针使用硅烷偶联剂的硅光学生物传感器

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

科研

生物工程

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

基质贴壁细胞的微管吸吮的估计单元刚度

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an area of productive research with over 1,400 published animal studies in just the last 5 years. Numerous techniques have been developed for quantifying smooth muscle activity of the stomach, small intestine, and colon. In vitro and ex vivo techniques offer powerful tools for mechanistic studies of GI function, but outside the context of the integrated systems inherent to an intact organism. Typically, measuring in vivo smooth muscle contractions of the stomach has involved an anesthetized preparation coupled with the introduction of a surgically placed pressure sensor, a static pressure load such as a mildly inflated balloon or by distending the stomach with fluid under barostatically-controlled feedback. Yet many of these approaches present unique disadvantages regarding both the interpretation of results as well as applicability for in vivo use in conscious experimental animal models. The use of dual element strain gages that have been affixed to the serosal surface of the GI tract has offered numerous experimental advantages, which may continue to outweigh the disadvantages. Since these gages are not commercially available, this video presentation provides a detailed, step-by-step guide to the fabrication of the current design of these gages. The strain gage described in this protocol is a design for recording gastric motility in rats. This design has been modified for recording smooth muscle activity along the entire GI tract and requires only subtle variation in the overall fabrication. Representative data from the entire GI tract are included as well as discussion of analysis methods, data interpretation and presentation.

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in rodents.

的微型双元应变计测量加工和植入<em&gt;体内</em&gt;胃肠收缩鼠害。

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires

A proper understanding of the force system created by various orthodontic appliances can make treatment of patients efficient and predictable. Reducing the complicated multi-bracket appliances to a simple two-bracket system for the purpose of force system evaluation will be the first step in this direction. However, much of the orthodontic biomechanics in this regard is confined to 2D experimental studies, computer modeling/analysis or theoretical extrapolation of existing models. The objective of this protocol is to design, construct and validate an in vitro 3D model capable of measuring the forces and moments generated by an archwire with a V-bend placed between two brackets. Additional objectives are to compare the force system generated by different types of archwires among themselves and to previous models. For this purpose, a 2 x 4 appliance representing a molar and an incisor has been simulated. An orthodontic wire tester (OWT) is constructed consisting of two multi-axis force transducers or load cells (nanosensors) to which the orthodontic brackets are attached. The load cells are capable of measuring the force system in all the three planes of space. Two types of archwires, stainless-steel and beta-titanium of three different sizes (0.016 x 0.022 inch, 0.017 x 0.025 inch and 0.019 x 0.025 inch), are tested. Each wire receives a single vertical V-bend systematically placed at a specific position with a predefined angle. Similar V-bends are replicated on different archwires at 11 different locations between the molar and incisor attachments. This is the first time an attempt has been made in vitro to simulate an orthodontic appliance utilizing V-bends on different archwires.

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires

The method presented here is designed to construct and validate an in vitro 3D model capable of measuring the force system generated by different archwires with V-bends placed between two brackets. Additional objectives are to compare this force system with different types of archwires and to previous models.

垂直 V 形弯曲力系统: 3D 弹性和刚性矩形矫正线的体外评价

A proper understanding of the force system created by various orthodontic appliances can make treatment of patients efficient and predictable. Reducing ...

Rat Model of Adhesive Capsulitis of the Shoulder

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other etiologies of comparable arthrofibrosis. The model includes extra-articular fixation of the shoulder in rats via scapular to humeral suturing, resulting in a secondary contracture without invading the intra-articular space and resulting in decreased rotational ROM and increased joint stiffness.
We used 10 Sprague-Dawley rats for the purpose of this study. Baseline ROM measurements were taken before glenohumeral immobilization. The rats were subjected to 8 weeks of immobilization before the fixation sutures were removed and changes in ROM and joint stiffness were evaluated. To evaluate whether immobilization resulted in a significant reduction in ROM, changes in kinematics were calculated. ROM was measured at each time point in the follow-up period and was compared to the baseline internal and external ROM measurements. In order to evaluate the stiffness, joint kinetics were calculated by determining the differences in torque (text and tint ) needed to reach the initial external rotation of 60&#176; and initial internal rotation of 80&#176;.
After the removal of the extra-articular suture fixation on follow-up day 0, we found a 63% decrease in total ROM compared to baseline. We observed continuous improvement until week 5 of follow-up, with the progress slowing down around a 19% restriction. On week 8 of follow-up, there was still an 18% restriction of ROM. Additionally, on follow-up day 0, we found the torque increased by 13.3 Nmm when compared to baseline. On week 8, the total torque was measured to be 1.4 &#177; 0.2 Nmm higher than initial measurements. This work introduces a rat model of shoulder adhesive capsulitis with lasting reduced ROM and increased stiffness.

This protocol presents an in vivo rat model of adhesive capsulitis. The model includes an internal fixation of the glenohumeral joint with extra-articular suture fixation for an extended time, resulting in a decreased rotational range of motion (ROM) and increased joint stiffness.

肩上粘 Capsulitis 大鼠模型的建立

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other ...

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture.
Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices.
This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

使用生物活性珊瑚基质提高解离神经细胞培养物的耐久性

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

移动式单分子FRET探头的自动化二维时空分析

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

用于使用受激拉曼散射显微镜进行延时活细胞成像的柔性腔室

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...