这项工作是从美国国立卫生研究院和优异评论RO1的C. - MT从弗吉尼亚州的研究服务，支持和个人NRSA到CWL

一般的设计考虑如果照片刺激的波长在可见光范围内，如optogenetic实验，的，系统的布局，将大大高于简单紫外光解实验。人们只需要购买一个双摄像头端口模块，显微镜公司所有。 DMD的平面，可以放置在任何一个的两个摄像头端口的共轭平面图像。但在紫外线光解从DMD的光必须被通过的外延照明路径（图3A），因为相机的成像管镜头是没有任何市售显微镜紫外线波长纠正。在350 nm的成像管镜头的球面像差是严重到足以防止能力为重点的光紧点，并产生足够的空间分辨率。这个问题是没有遇到因为管取下镜头时沿同一光轴准直激光束带来，在激光共聚焦显微镜。因为管镜片也有部分在紫外线波长的球面像差纠正所带来的问题是可以解决的紫外光照射在通过外延荧光路径。我们可以扩展光学显微镜在任何程度杂志的愿望。科学界一般快速在这个主题感兴趣，并有许多来源，教得好。 为了尽量减少显微镜的修改，因为紧凑的空间限制，在落射荧光装置，DMD是放置在一个新创建的共轭影像平面创建一个中继镜头（相对于点出的DMD单元的位置显微镜）。商业紫外线纠正继电器镜头购（专业光学）（点图和显微镜）。 的DMD的照明微镜可以在收到其二进制输入一个积极的和负12 °倾斜，相对于平面芯片之间切换。旋转轴是沿对角线角落每个镜像（45 °芯片的两侧）。镜像旋转的方向是指定的黄金在芯片表面上的一个角落的彩色三角形。微镜的倾斜角度决定传入照明光束的音调和azithmus对齐。照明光束的音调，需要24 ° DMD芯片和azithmus需要镜子旋转轴垂直的垂直轴。精确的光束对准高效运行的关键。在原型系统中，我们设计了多个手动倾斜调整，使我们能够正确的设计和加工的不足之处。这导致在一个系统中，是相当多的笨重的比是必要的。替代的，更紧凑带来光的安排是可能的（如果认为有用，显示替代设计）。 对于光解实验，激光源产生的高光强度聚焦光束快速uncaging必要。 optogenetic不需要高强度急剧聚焦光线的实验，一个非相干光源就足够了。在这里描述的光解实验，我们采用一个准连续二极管泵浦固态（DPSS）的频率增加了两倍NdVO4激光（1瓦，355纳米）。 （光解光源的选择，对于一个详细的讨论见参考文献2）。因为只有一个激光输出的一小部分是实际交付的标本时使用的DMD系统，需要一个相对高功率激光在这里所描述的实验。传递到试样的光量成正比的激光束照射的区域内的ON / OFF的镜子比。 如果激光光源是必需的，它是最好的发射到多模光纤激光的输出，所以，它可以容易沿着正确照亮DMD的轴的地位和的导向。光通过光纤传输解决第二个问题，如何消除斑点相干照明的固有模式。该纤维是伤口周围的压电纤维担架“（型号915，加拿大的仪器及研究有限公司）〜40千赫的振荡。 （点出在这些钻机）纤维的微观足够的移动散斑毫秒为单位的持续时间在每张照片的刺激脉冲模式多次，从而有效地消除斑点的效果。光纤输出，然后平行与紫外显微镜物镜（奥林巴斯DApo20UV）。 （指出这一点）校准系统的光学分辨率，如图所示。 3B。有效的生理功能的现场照片作为位置测量电流的流动幅度的决议如图刺激。 3C。对不同强度photostimulation的电流响应如图所示。 3D。 系统的操作： 联合登记与CCD像素的DMD反射镜在内部软件的书面决定个别成像CCD相机的具体像素的DMD反射镜对应。然后在这个软件的图形用户界面（GUI），允许用户指定对应于该地区的鼠标标签的CCD图像的DMD反射镜。因此，照片刺激的位置，可以用来标记只需对计算机屏幕上显示的图像的光标移动和点击标签地区的利益。 （树突状乔木的荧光图像将显示在电脑屏幕上，学生将在计算机屏幕上的形象标志的一些地点。） 编程光交付模式由操作员的电脑屏幕上标出的模式是一系列作为一个单独的图像存储。每个空间格局的激光脉冲的时间，然后到这是与数据采集程序（pClamp）集成的软件编程。 （说明这一点）。 协调的激光脉冲的DMD模式数据采集​​程序发起和协调的DMD电子，激光浇注，和收购的补丁的时间夹住目标神经元的电信号。 （在计算机上模拟这个顺序） 实验数据： 在远端树突状的分支点的非线性求和与电极的树突状融合传统上一直进行离散位置的不同幅度的刺激，而不是扩大面积的同时，保持恒定强度的刺激。 electrogenesis和依赖渠道的位置招聘饱和有关的问题可以影响的结果和结论。分布式树突状刺激可以很容易地使用一个DMD为基础的系统（图4A）。 photostimulation点的数量增加投入力度是不同的。我们可以看到，可以跨越空间的总和分支点越来越成为到达的两个女儿分行加大投入幅度超线性非线性。超线性主要是介导NMDA受体，而不是电压门控通道（图5） 代表性的成果<img src="/files/ftp_upload/2003/2003fig1.jpg" alt="图1" /> 图1：在DMD微镜的运作。 （侧视）针对个别镜子的静电引力导致两个可能的方向从横向12 °倾斜的镜子。在ON位置时，入射光的指示沿垂直于光轴的芯片。在OFF位置，入射光是针对48 °离轴。光打薄的镜子之间的差距是针对24 °离轴。 （顶视图）的倾斜是面向45 °两侧的反光镜和芯片。 <img src="/files/ftp_upload/2003/2003fig2.jpg" alt="图2" /> 图2。现代的荧光显微镜的基本布局。过滤器的多维数据集是定位在“无限空间”的目标和管镜头之间。是导致相机的成像路径，有一个样品带来的光的照明路径。这两个光路的管镜头通常是在设计和焦距的不同。照明，但不成像，管镜头的目的是在紫外光谱的操作。 <img src="/files/ftp_upload/2003/2003fig3.jpg" alt="图3" /> 图3。紫外线基于DMD的系统布局。如果照明是在紫外光谱，那么它必须通过管镜头，纠正紫外线带来。管镜头在成像路径是不是设计的紫外线。一个中继系统是需要创造一个方便的共轭平面图像。不同的共轭显微镜图像平面标志是红色虚线的线条。预计在其他的共轭平面在一个平面图像的明亮模式。在成像过程中，在样品平面图案投射到探测器/相机。由DMD生成的模式用于照明投射到样品。 <img src="/files/ftp_upload/2003/2003fig4.jpg" alt="图4" /> 图4。可见光DMD投影系统的布局。如果在可见光范围内的照明灯，它是可能带来的DMD产生通过成像光路的光纹。这可以方便地实现与commerci人的双摄像头端口附件和相应的分色镜。 <img src="/files/ftp_upload/2003/2003fig5.jpg" alt="图5" /> 图5系统的横向分辨率。 （一）荧光靶上的光学分辨率为1〜2微米。 （b）有效解决跨枝蔓测量光解笼谷氨酸〜5微米。距离增加是由于结合谷氨酸和扩散的有限宽度的枝蔓。 （三）光解可以模仿的突触活动的动力学。家庭的电压钳位电流的反应，是因为在交付的枝蔓光能量的变化。 <img src="/files/ftp_upload/2003/2003fig6.jpg" alt="图6" /> 图6。非线性跨树突分支点的总和。 （一）适用于分布式输入两个树突分支分开。他们各自的电压响应如下所示。 （二）刺激，然后同时进行。测量响应（红色曲线）是不同的两个个体反应（灰色跟踪）的算术总和比。 <img src="/files/ftp_upload/2003/2003fig7.jpg" alt="图7" /> 图7。Supralinear求和是NMDA受体依赖性。 （一），有选择性的NMDA受体拮抗剂APV块supralinear的总和。 （二）绘制刺激强度的关系及其NMDA受体拮抗剂的敏感性。

<ol>
	<li>Scanziani, M., Hausser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiology+in+the+age+of+light.">Electrophysiology in the age of light.</a> Nature. 461, 930-939 (2009).</li><li>Tang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolysis+of+caged+neurotransmitters:+Theory+and+procedures+for+light+delivery.">Photolysis of caged neurotransmitters: Theory and procedures for light delivery.</a> Curr. Prot. Neurosci. , 6.21.1-6.21.12 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+Technology+Feature,+Cell+imaging:+Light+activated.">Nature Technology Feature, Cell imaging: Light activated.</a> Nature. 456, 826-827 (2008).</li><li>Lutz, C., Otis, T. S., DeSars, V., Charpak, S., DeGregorio, D. A., Emiliani, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Holographic+photolysis+of+caged+neurotransmitters.">Holographic photolysis of caged neurotransmitters.</a> Nature Methods. 5, 821-827 (2008).</li><li>Hornbeck, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Digital Light Processing and MEMs: An overview. , .</li></ol>

基于DMD的photostimulation方法的优点是最明显的目标中占有相对大面积的情况下。如果利益的目标是非常小的，如几个树突棘，的，连续扫描共聚焦和双光子系统可能是更好的方法。 DMD的方法的一个重大弱点是其低效利用现有的光。大部分可用的光必然是向关镜，不使用。 基于DMD的制度是最适合在可见光范围内操作。我们期望与光遗传学实验时，DMD的基础photostimulation系统将作?...

Erratum: Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points

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<td>Modern upright fluorescent microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>CCD camera and image acquisition software</td>
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<td>Computer and data acquisition/interface system</td>
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<td>DLP Discovery Developer Kit </td>
<td>&nbsp;</td>
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<td>ALP3 USB interface</td>
<td>&nbsp;</td>
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<td>S2 + Optics w/LED</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Dual camera port unit</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>355nm frequency tripled NdVO4 laser (~1 W)</td>
<td>&nbsp;</td>
<td>DPSS Laser Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Laser shutter Model LS6</td>
<td>&nbsp;</td>
<td>Uniblitz</td>
<td>&nbsp;</td>
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<td>Multimode optical fiber and fiber stretcher Model# 915</td>
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<td>Canadian Instrument and Research, Ltd </td>
<td>&nbsp;</td>
<td>100 um core multimode fiber</td>
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<td>Multimode Fiber launcher </td>
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<td>Oz Optics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Signal generator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>up to 50 kHz</td>
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<td>Beam collimator</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>DApo20UV340</td>
<td>&nbsp;</td>
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<td>UV relay lens</td>
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<td>Special Optics</td>
<td>#: 54-25-60-355</td>
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patterned photostimulation with digital micromirror devices to investigate dendritic integration across branch points

Light是一个通用和精确的手段来控制神经元的兴奋性。近期出台的通道的视紫红质和笼中的神经递质，如光敏感效应，导致以利益在开发更好的手段来控制光的模式，在空间和时间是有用的实验神经科学。一个传统的战略，在共聚焦和双光子显微镜就业，是集中光线的衍射有限点，并在感兴趣的区域，然后扫描顺序单点。这种方法就出现了问题，如果大面积必须在很短的时间窗口，一个问题比成像更适用photostimulation的刺激。另一种方法是数字微镜器件（DMD）的援助项目的完成对目标的空间格局。 DMD的做法是有吸引力，因为硬件组件都相对便宜，而且是由商业利益的支持。因为这种制度是不是直立式显微镜，我们将讨论在这样一个DMD系统的建设和运营的关键问题。即使我们将主要描述紫外光解的制度建设，为建设optogenetic实验可见光系统简单得多的修改也将提供。紫外光解系统是用于结转库存实验，以研究在神经科学中的一个根本性的问题，如何在空间上分布在远端树突分支点综合投入。结果表明，一体化非线性跨分支点和supralinearity主要是由NMDA受体介导的。

Watch this Scientific Journal Video about Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points at JoVE.com

Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points

数字微镜器件（DMD），可以在时间和空间产生复杂的图案，用以控制神经元的兴奋性。的DMD系统的设计，施工，操作的有关问题进行了讨论。这样一个系统，使在远端树突分支点非线性一体化的示范。

图案与数字微镜装置Photostimulation调查跨分支点的树突状融合

Light is a versatile and precise means to control neuronal excitability. The recent introduction of light sensitive effectors such as channel-rhodopsin ...

patterned-photostimulation-with-digital-micromirror-devices-to

Research

JoVE Journal

Bioengineering

15.5K 次观看.  University of Maryland School of Medicine. 数字微镜器件（DMD），可以在时间和空间产生复杂的图案，用以控制神经元的兴奋性。的DMD系统的设计，施工，操作的有关问题进行了讨论。这样一个系统，使在远端树突分支点非线性一体化的示范。

视频: Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

模型和方法来评价药物输送系统在整个细胞屏障的运输

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

平面梯度扩散系统的调查趋化三维胶原基质

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

制造棉分析仪器

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

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 ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

实验和有限元议定书调查中性和带电溶质的跨关节软骨运输

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable microfluidic devices. Here, a protocol for the fabrication of plastic microfluidic devices is demonstrated, where soft lithographic techniques are used to embed transparent Calcium Fluoride (CaF2) view-ports in connection with observation chamber(s). The method is based on a replica casting approach, where a polydimethylsiloxane (PDMS) mold is produced through standard lithographic procedures and then used as the template to produce a plastic device. The plastic device features ultraviolet/visible/infrared (UV/Vis/IR) -transparent windows made of CaF2 to allow for direct observation with visible and IR light. The advantages of the proposed method include: a reduced need for accessing a clean room micro-fabrication facility, multiple view-ports, an easy and versatile connection to an external pumping system through the plastic body, flexibility of the design, e.g., open/closed channels configuration, and the possibility to add sophisticated features such as nanoporous membranes.

A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described.

生产塑料微流控装置与查看端口对可见光和红外透明软光刻过程

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: 一种功能强大、无创的研究细胞能量代谢的方法

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

High-resolution Patterned Biofilm Deposition Using pDawn-Ag43

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms into arbitrary spatial patterns at high spatial resolution. The technique uses a genetically encoded optogenetic construct&#8212;pDawn-Ag43&#8212;that couples biofilm formation in E. coli to optical stimulation by blue light. We detail the process for transforming E. coli with&#160;pDawn-Ag43, preparing the required optical set-up, and the protocol for culturing patterned biofilms using pDawn-Ag43 bacteria. Using this protocol, biofilms with a spatial resolution below 25 &#956;m can be patterned on various surfaces and environments, including enclosed chambers, without requiring microfabrication, clean-room facilities, or surface pretreatment. The technique is convenient and appropriate for use in applications that investigate the effect of biofilm structure, providing tunable control over biofilm patterning. More broadly, it also has potential applications in biomaterials, education, and bio-art.

We demonstrate a method for depositing Escherichia coli bacterial biofilms in arbitrary spatial patterns with a high resolution using optical stimulation of a genetically encoded surface-adhesion construct.

使用 pDawn-Ag43 的高分辨率图案生物膜沉积

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms ...