<p class="jove_content"我们感谢精辟的讨论DD张，并慷慨地提供试剂。 MJ是支持由美国国立卫生研究院心血管培训资助，亚利桑那州的科技研究计划基金，学院科学家的成就奖励。这项工作是由美国国立卫生研究院主任的新的创新奖（1DP2OD007161 01），美国国家科学基金会（0855890），詹姆斯S ·麦克唐纳基金会的支持。</p

1。创作的图案使用的模具<ol><li>概念设计的图案。创建模式，有助于形成细胞网络，控制细胞的接触，分离细胞，模仿自然的结构，或以其他方式指导细胞粘附和安置。 </li><li>计算机计算机辅助设计或CAD的光掩模的模式和创造的基础创造。所需的图案设计CAD软件如AutoCAD，然后将其发送给外部公司生产光罩。 </li><li>光刻生产主模式。玻片是一个薄薄的正面抵抗或厚厚的消极抵制取决于正在创建的结构尺寸与图案。玻片用于其好处是成本低，比硅片强，而不是生产尽可能多的灰尘，当断。另外，其他方便的结构，如衍射光栅，可作为主图案。 </li><li>图案的幻灯片是一叠没有图案的幻灯片。所有步骤都执行一个干净的流罩内，以尽量减少灰尘。 </li><li>创建主模具。锡箔比一叠幻灯片略大一些的容器创建持有30克的聚二甲基硅氧烷（PDMS）中，然后倒在光刻图案的表面，以创建一个初始的模具。 PDMS的是脱气，并允许一到两天治愈。 </li><li>一旦固化的PDMS揭下主模式和形式，为下一步的一个模具。 </li><li> 6 G是2部分的环氧树脂（DEVCON＃14310（6克）的混合1分钟，然后倒入母模，脱气，可以治愈。脱气环氧树脂必须迅速完成（&lt;8分），使用了很多的泡沫破周期使用之前，它只是薄薄的一层环氧套和泡沫去除，就不可能消除气泡。6克用于生产薄薄的一层，这有利于快速的泡沫去除。 </li><li>一个小时后，第2部分的环氧树脂将部分治愈更多的环氧树脂可以在上面增加而脱气产生较厚的结构，这是easer在以后的步骤处理。环氧树脂是可以治愈一到两天。 </li><li>一旦固化，环氧树脂被删除从PDMS母模和胶带包裹，形成一个容器周围的环氧树脂模具。然后创建一个工作模浇注到环氧树脂模具硅橡胶，脱气的PDMS，并固化为一到两天。 </li><li>一旦固化，模具的工作是剥落的环氧树脂和储存，以保持清洁。 </li></ol> 2。表面图案与细胞指导模具<ol><li>模具工作的小部分被切断，并放置到聚苯乙烯培养皿。这些模具部分的位置标记。 </li><li>形成了从三脚架的小部分和加权，以确保形模具之间的工作和在培养皿表面接触。盘底部可观察到安置好。 </li><li>大会是放入等离子室。等离子体处理是发起并持续在29.6瓦（最大功率）10分钟使用空气等离子在150帕斯卡。 </li><li>等离子体处理后，模具和重量提审都被删除。 </li><li>在培养皿放在紫外光下10分钟内的生物安全柜消毒和储存在那里，直到种子细胞。 </li></ol> 3。播种与细胞表面<ol><li> CRL - 2266的SH - SY5Y人神经母细胞瘤或其他细胞培养的细胞类型中使用标准协议。 </li><li>合流的SH - SY5Y的CRL - 2266人神经母细胞瘤细胞测量视觉上的图案的表面上，播种前。 </li><li>标准的细胞分裂是开展以消除他们的培养皿的表面细胞。 </li><li>图案培养皿的表面上的细胞，一旦自由浮动，种子被摊薄后达到预期的汇合时，图案的表面上放置。 </li><li>这些细胞被允许定居和附着于表面。 </li><li>图案的培养皿中培养几个小时到几天，而细胞聚集到血浆创建的模式。 </li><li>当培养的神经母细胞瘤，维甲酸（10μM）是每天补充，以诱导细胞分化成神经元样的状态。维甲酸此外，在黑暗中。 </li><li>维甲酸是添加了好几天，每天分化后，将观察到的。 </li><li>媒体是取代每隔3-4天。 </li></ol> 4。观察和分析<ol><li>随着时间的推移，以观察细胞的行为采取定期的活细胞或视频图像。对于实时图像，细胞置于显微镜舞台顶部保持在37℃，湿度为100％，5％的CO 2细胞的孵化器。细胞也可以固定和染色在荧光下观察</li><lI&gt;细胞已被抓获的图像或视频导入到图像分析系统，测量的速度增长，细胞的大小，迁移速度，分化，对齐方式，特定的蛋白质的位置，以及其他包括开展。 </li></ol> 5。代表性的成果： 实施等离子体光刻技术的一个典型的结果是形成一个细胞，这些细胞类似于一些任意的或自然的结构模式。这被认为是在图1A - B线和神经元的网络已经建立。可用于其他类型的细胞， 以及图1C - D，这说明人脐静脉内皮细胞（HUVEC）和C2C12骨骼肌细胞形成电网。材料，如聚- L -赖氨酸，也可以是图案，图1e，以方便某些类型的细胞和作其他用途的附件。在显示神经元的情况下，创建的细胞，它们之间的连接网络。这复制在大脑中神经元之间的离散连接邻近的细胞，从而影响大脑的运作的自然会发生什么。随着这种结构在实验室的创建，连接的数量，位置，频率和其他因素，可以得到系统的控制。这些传讯的结果可直观地测量和额外投入，如化学或电刺激可实施探测网络的行为。 一个消极的结果将是一个杂草丛生的或不完整的的模式或受污染的样品。不完整或杂草丛生的模式会导致要么太少或太多的细胞不会提供一个理想的细胞量的格局，从播种基材。此外，如果不正确的设计模式（例如，线条过于狭窄）的细胞不会被它成功地附着和生长。在受污染的样品情况下，适当的清洁不会一直保持在步骤之一，但适当的细胞培养协议，由于血浆图案也消毒基板时，这是罕见的的。 <img src="/files/ftp_upload/3115/3115fig1.jpg" alt="图1"/> 图1。细胞和蛋白质的图案。

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这里介绍的调查方法使细胞的多细胞网络，模仿生物结构以及复杂的图案创作提供了一种方法来产生刺激的性质，然后方便两个分组的细胞的行为和环境因素的单细胞反应的调查亚细胞。使用这种方法是简单而强大的的，在相同的细胞培养实验室，因为它可以迅速地进行低成本设备。这也是强烈的细胞敏感，可以便于观察由此产生的行为和性质稳定的长的时间周期，这使得长期的细胞行为的调查。此外，它允许多元化的，因为它是兼容多种细胞类型，并可以创建任意模式进行的实验。该技术的长期稳定派生从表面功能的血浆传授，是表面的一部分，而不是涂层或其他层，它可以被删除或降级的事实。如果保存液下，这种类型的修改，可以保留其细胞数月的指导能力。 必要的实验，以确保有意义的结果，最关键的部分是创建主模式，最终将被用于细胞指导。如果这种模式是不正确的设计，细胞不会妥善应对的格局，并可能不会产生有益的行为。如线的宽度，图案间距，和其他的参数，可以大大影响与特定的模式的细胞相容性，通常是这样的参数的范围，可以筛选最合适的设计初始光掩膜创建。 出图案的账面相关的其他重要参数包括模具创造，保持无尘环境中，细胞种植和细胞培养一般不育。可透过各种传输采取步骤，以允许重复的PDMS铸造环氧树脂模具模具创造。环氧树脂模具创建，因为它不会以同样的方式降低作为抵制与小，高宽比结构，根据反复铸造模具。如果做得正确，传递模塑的尺寸和产量不会受到影响，但如果做得不正确，如脱气很差，固化不完全，或过度加热，气泡，表面粗糙度和模式的可发生变形，从而影响最终的结果。关于保持无尘环境，模具必须保持尽可能干净的任何灰尘会干扰工作的PDMS模具和表面，从而适当的等离子体屏蔽和化学图案之间的适当联系。细胞的播种密度，还必须进行优化，以确保既不太少或太多的细胞居住格局区域必须保持无菌，以避免细菌和其他污染所使用的细胞。 该技术也可以被纳入与其他元素，如微流体，微电极，和机械探头。这提供了额外的刺激细胞，为了更好地复制实验和今后的工作过程中的各种生理条件，重点是学习这些组合的效果

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>等离子体清洁和真空室</td><td> Harrick等离子</td><td> PDC - 001 </td><td></td></tr><tr><td>倒置荧光相差显微镜</td><td>尼康</td><td> TE2000 - U </td><td></td></tr><tr><td>显微镜阶段顶部孵化器</td><td> AmScope </td><td> TCS - 100型</td><td>修改为包括一个外壳，提供一个合适的气氛</td></tr><tr><td>聚苯乙烯培养皿</td><td> VWR </td><td> 25384-090 </td><td></td></tr><tr><td>聚二甲基硅氧烷（PDMS） </td><td>道康宁</td><td> Sylgard 184 </td><td></td></tr><tr><td>环氧树脂</td><td> DEVCON </td><td> 2吨明确环氧＃14310 </td><td></td></tr><tr><td>细胞培养基</td><td>各种</td><td></td><td>媒体如下细胞类型的标准配方</td></tr><tr><td>维甲酸</td><td>西格玛</td><td> R2625 </td><td></td></tr></tbody></table>

plasma lithography surface patterning for creation of cell networks

与微型和纳米级分辨率的细胞微环境的系统操作，往往需要破译各种细胞和分子的现象。为了满足这一要求，我们开发了一种等离子体的光刻技术来操纵创造与功能，范围从100纳米到毫米大小的图案表面的细胞微环境。这项技术的目标是可以学习的，在一个可控制的方式，单个细胞的行为，以及细胞和它们之间的相互作用的群体。 这种等离子体光刻方法的基础上选择性的表面化学修饰对基材通过屏蔽接触低温等离子体物理模具。这种选择性的屏蔽叶化学模式可以指导细胞粘附和运动。这种模式，或表面的模板，然后可以用于创建细胞，其结构可以模仿自然界中发现的的，并产生一个可控环境实验研究网络。该技术以及适合研究生物现象，因为它产生的生物相容性的方式透明的聚合物基材的稳定的表面图案。表面图案的最后几个星期到几个月，从而引导长的时间周期，这有利于长期的细胞过程，如分化和适应，研究细胞的相互作用。表面改性主要是化学性质，因此不引入地形或物理干扰结果的解释。它也不会涉及任何恶劣或有毒物质来实现图案和组织文化的兼容。此外，它可以应用于不同类型的聚合物基板，由于能力调整自己的属性是理想的，被广泛用于生物应用修改。分辨率可以实现也是有利的，如迁移，附着力强，或有约束力的具体过程的隔离允许离散明确的意见，在单一到多节水平。 该方法已涉及迁移，信号，组织的形成，和网络的行为和神经元的相互作用，在提审调查，形成不同网络的不同类型的细胞。

Watch this Scientific Journal Video about Plasma Lithography Surface Patterning for Creation of Cell Networks at JoVE.com

Plasma Lithography Surface Patterning for Creation of Cell Networks

一个多功能等离子体光刻技术已经发展到稳定的表面图案为指导细胞附着生成。这种技术可用于创建网络，包括那些已用于研究几种，不同的细胞类型，模仿自然的组织和细胞。

等离子体表面图案光刻技术的蜂窝网络创作

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

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12.6K 次观看.  University of Arizona. 一个多功能等离子体光刻技术已经发展到稳定的表面图案为指导细胞附着生成。这种技术可用于创建网络，包括那些已用于研究几种，不同的细胞类型，模仿自然的组织和细胞。

视频: Plasma Lithography Surface Patterning for Creation of Cell Networks

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

科研

生物工程

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

在光刻技术定义的聚对二甲苯-C细胞图案：二氧化硅<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

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 ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

活细胞层的快速消减带图案微流控探头

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&amp;#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

Scaled Anatomical Model Creation of Biomedical Tomographic Imaging Data and Associated Labels for Subsequent Sub-surface Laser Engraving (SSLE) of Glass Crystals

Biomedical imaging modalities like computed tomography (CT) and magnetic resonance (MR) provide excellent platforms for collecting three-dimensional data sets of patient or specimen anatomy in clinical or preclinical settings. However, the use of a virtual, on-screen display limits the ability of these tomographic images to fully convey the anatomical information embedded within. One solution is to interface a biomedical imaging data set with 3D printing technology to generate a physical replica. Here we detail a complementary method to visualize tomographic imaging data with a hand-held model: Sub Surface Laser Engraving (SSLE) of crystal glass. SSLE offers several unique benefits including: the facile ability to include anatomical labels, as well as a scale bar; streamlined multipart assembly of complex structures in one medium; high resolution in the X, Y, and Z planes; and semi-transparent shells for visualization of internal anatomical substructures. Here we demonstrate the process of SSLE with CT data sets derived from pre-clinical and clinical sources. This protocol will serve as a powerful and inexpensive new tool with which to visualize complex anatomical structures for scientists and students in a number of educational and research settings.

Scaled Anatomical Model Creation of Biomedical Tomographic Imaging Data and Associated Labels for Subsequent Sub-surface Laser Engraving SSLE of Glass Crystals

A methodology is described herein for representing anatomical imaging data within crystals. We create scaled three-dimensional models of biomedical imaging data for use in Sub-Surface Laser Engraving (SSLE) of crystal glass. This tool offers a useful complement to computational display or three-dimensionally printed models used within clinical or educational settings.

缩放生物医学层析成像数据的解剖模型的创建和相关联的标签玻璃晶体的随后的子表面激光雕刻（SSLE）

Biomedical imaging modalities like computed tomography (CT) and magnetic resonance (MR) provide excellent platforms for collecting three-dimensional data ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

基于多步法链位移光刻的基因功能表面固定化研究

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...