作者要感谢道格拉斯博士戈茨（化学系和生物分子工程，俄亥俄大学）和费边Benencia博士（生物医学俄亥俄大学系）对有见地的讨论和审稿。我们也感谢克里斯托弗Huppenbauer博士的有益的技术讨论（W. Nuhsbaum公司）。这项工作是由美国国立卫生科学基金会（CBET-1106118）和国家研究院​​（1R15CA161830-01）资助。

1。软件安装<ol><li>购买StreamPix 5多摄像头软件SimulPix模块或能够收集并结合了黑白CCD相机拍摄的图像的其它图像软件。 </li><li>对于StreamPix 5最低要求包括：搭载Core 2 Duo处理器，2.4 GHz或更好地与2 GB RAM或更高的PC。受支持的IEEE数字或模拟相机和兼容图像采集卡。窗户XP/7/Vista 32位或64位。监视器支持分辨率1024×768或更高。 （建议OCU表达16倍或更高）的图形适配器具有良好的2D性能和7,200 RPM的硬盘驱动器，用于记录与RAID-O配置。 </li></ol> 2。安装双摄像头系统能够双色同步实时成像<ol><li>通过滑动DC2 C型安装适配器插入MICR的视频端口连接DC2的配光发射分离器或类似的双摄像头发射分光装置，在显微镜OSCOPE，使得设备上的二向色性的外壳是可访问。 </li><li>插入两个黑白CCD摄像机转化为雌C接口1和女C型2。相同的摄像机是优选的。类似的相机也可使用，但兼容性是依赖于硬件的内部，最重要的是CCD图像传感器。 </li><li>使用图像处理软件来显示来自摄像机的图像。下面的步骤（2.3.1 - 2.3.4）适用于StreamPix 5与SimulPix模块，并且必须适用于其他的多摄像头成像软件。 <ol><li>打开StreamPix 5和任务色带选择“工作空间”。 </li><li>打开“工作区管理器”位于最右边的屏幕并选择“新工作区”。 </li><li>命名一个摄像头后，按照软件提示选择从预填充的列表中选择一个抓取。选择抓取匹配的相机型号和重复的第二个摄像头的工作区。对于合并后的工作空间，重复一次以上，但“所有的相机都在使用”错误米essage将出现。此消息是正常的。 </li><li>一旦合并后的工作区被加载时，分配摄像机从工作区1和2的工作区，以在对接面板位于上观看屏幕的右侧合并的工作区。 </li></ol></li><li>在双摄像头发射分光系统对准摄像头。 <ol><li>在明视场或通过使用PlanApo 40X物镜（或更大）的制造商所提供的校准网格相衬对准摄像机。 </li><li>发送光显微镜目镜，将被提供由制造商在显微镜舞台上的金属涂层校准网格，方形网格上的显微镜载物台。 </li><li>使电网成为关注的焦点。 </li><li>取代，但不删除，分色住房对准摄像头1（旁路相机）。显示网格不分级，不自动缩放。在使用C型底座端口1的对焦调节旋钮聚焦影像。 </li><li>推分色住房进入双channe升获得充分，从而图像被分割为二向色到两个相机设备。 </li><li>松开3 5/64“设定螺钉和转动在阴C型2第二相机直到网格的取向是相同的两个图像中，利用聚焦调整拨盘上的C型安装口2，从相机带来的图像2成为关注的焦点。 </li><li>使用组合图像中的成像软件的合并工作区完成对齐。这允许人们看到的校准网格的两个图像的实时伪覆盖。只使用R / L旋钮右侧DC2的调整合成图像的位置。调整此旋钮，直至图像的左/右垂直边界的精确对准。 </li><li>使用U / D转换旋钮调整第二个图像的上/下排列，直到水平网格条正确对齐。 </li><li>如果在向上/向下对准轻微旋转摄像头松开三个5/64“英寸SCRE发生时，正确的这个问题WS相机2和旋转，直到显示的图像正确对齐。 </li></ol></li></ol> 3。对平行平板流动腔附着力测定制剂癌细胞<ol><li>培养的BT-20乳腺癌细胞在最低必需培养基（MEM），补充以完成介质具有在37℃，10％胎牛血清（FBS）和1％青霉素-链霉素和CO 2如前所述2 5.0％。 </li><li>嘉实癌细胞用稀胰蛋白酶处理，并计算细胞血球。 </li><li>洗涤和悬细胞于10 7个细胞/ 0.1％白蛋白的牛血清（BSA）的DPBS中的钙和镁（DPBS +）毫升。 </li><li>稀释的第一抗体，纯化的小鼠抗人CD24和纯化的大鼠HECA-452（抗-sialofucosylated抗原），在0.1％BSA的DPBS +预先确定的最佳浓度。孵育细胞30分钟，9，10。 </li><li>离心细胞在1,200 rpm下，得到的细胞沉淀。 ASPI评价第一抗体，并用0.1％BSA的消毒副产物+洗涤细胞两次。 </li><li>稀释的二抗，山羊抗小鼠IgG AlexaFluor 568（发射最大值，603纳米）和山羊抗大鼠IgM AlexaFluor 488（发射最大值，519纳米），以预先确定的最佳浓度在0.1％BSA的DPBS +。孵育细胞30分钟。 </li><li>以10 6细胞/ 0.1％BSA的DPBS +毫升洗涤细胞两次并悬浮在0.1％BSA的DPBS +。 </li></ol> 4。对平行平板流动腔附着力测定制剂反应底物<ol><li>连接两个独立的管道长度，以适合在35 mm组织培养皿内的平行平板流动腔室的入口和出口端口。一个管将连接到标记的细胞悬浮于0.1％BSA的DPBS +和其它的注射器泵，这将在指定的流量抽出流体的贮存器。 </li><li>连接真空管线到流动室1。 </li><li>定位流室在35mm组织培养二SH含中国仓鼠卵巢细胞单层表达E-选择素（CHO-E）。 CHO-E细胞在MEM完全培养基中，在37℃和5.0％CO 2的2。 </li><li>调整流动室和/或流室垫圈，以确保良好的真空密封件1。 </li><li>从水库抽取液体，监测流动室组件进行适当的密封与 ​​空气形成气泡1没有视觉证据。 </li><li>将流动室组件上的显微镜（Leica DMI 6000B）1。 </li></ol> 5。双色图像采集<ol><li>电力徕卡EL-6000紧凑型光源，或类似的装置，以产生高强度光夫妇到一个光导。使用倒置显微镜通过组合滤块， 即 G / R（绿色/红色）过滤器的立方体通过这个光，激发所需的颜色/亮度的荧光。 </li><li>确保分色住房是在正确的操作姿势（郁闷）和是不是在旁路模式。 </li><li>操作显微镜的荧光模式，并选择适当的荧光滤块（绿色/红色）。 </li><li>确定曝光时间和增益摄像头1（红色; 620±60 nm）和摄像机2（绿; 535±40纳米）。 <ol><li>踩下分色同时在红色和绿色发射渠道获取的图像。使用带有标记的细胞只有红色荧光的流动室试验中，通过调整摄像机1的曝光时间在成像软件的“实时设置”获得图像中的红色通道。 </li><li>相机2的曝光时间匹配到照相机1的曝光时间。如果图像中的绿色通道观察，渗出通过伪影的存在，需要在两个相机的曝光时间可以缩短。 </li><li>保持曝光时间相当于在摄像机1和摄像机2，减少曝光时间，直到扩散到工件不再在绿色通道可见。调整摄像机1的增益，以提高图像visibilitY的红色通道，如果必要的。 </li><li>重复步骤5.4.1 - 5.4.3与标记的细胞只有绿色荧光，但与照相机2限定的曝光时间，以图像单元中的绿色通道而不在由照相机1所收集的红色通道渗出通过工件。 </li><li>返滴定抗体，如果放气通过伪影不能被消除11，或者如果摄像机1和摄像机2的曝光时间是显着不同，使得合并后的图像可以是在时间上对齐。 </li></ol></li><li>使用图像处理软件来查看从流室实验二黑白CCD摄像机同时拍摄的图像。 </li><li>合并使用Streampix 5的SimulPix模块或另一种软件包，这两个摄像头采集到的图像。 </li><li>使用SimulPix或替代软件数字校正调整，如有必要，在空间上对齐拼接的图像，。图像可以用SimulPix模块为水平，垂直纠正和旋转调整。 </li></ol>

<ol>
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作者宣称，他们有没有竞争的财务权益。

双摄像头发射分光系统需要在应用中的细胞或分子的运动是快速捕捉高品质图像的空间，时间，以及光学分辨率。在产生代表性的结果，在双相机发射分光系统，包括软件的设置，相机设置的参数，进行优化以获得合并的图像序列，其中轧制和贴壁细胞，空间和时间上对齐。此优化步骤是关键的，因为未对准可导致图像不正确传达所研究的物理现象， 例如 ，观察到滚在两个绿色和红色通?...

方法分子在细胞表面上，如免疫染色分析，采用被化学偶联至荧光基团，从而允许检测目标分子的探针。活细胞成像和流体动力流动为基础的细胞黏附测定法通常记录的黑白CCD相机设计在细胞和/或分子水平1，2捕获的生理过程。这些摄像机是高度敏感的，提供快速的帧速率（大于每秒30帧），和（由于快速的帧速率和较短的曝光时间），提供出色的时间分辨率。然而，黑白相机只能捕捉到一个单一的发射通道（检测单个荧光基团）来采集图像。单个相机发射分光系统可并入到捕获多个发射信道，但往往减少视场并需要相同的曝光时间进行摄像的所有通道。要抓紧细胞标记MULTIP的全彩色光谱勒荧光团，一个彩色照相机可以作为一种替代方法。然而，彩色相机通常不能够提供期望在某些应用中的活细胞成像的时间分辨率。另一个成像装置所需的应用中，有利的是，图象的活细胞在多个波长，同时保持高的时间分辨率。一个主要的实验应用是在平行平板流动腔粘附试验，其中细胞被灌注在生理学相关条件下在一个潜在的反应基片1，3。在流动细胞表达特定细胞表面分子可能粘附和轧辊的基体如表达粘附分子或表面吸附的细胞外基质蛋白4，5的细胞单层上。轧制细胞可以在几分之一秒进行旋转和平移运动。上的滚动和粘附的细胞，如簇的细胞表面分子的分子功能，还具有电位人经过积极的重组在细胞表面上。因此，成像系统必须提供一个特殊的时间分辨率（每秒或更高30帧和“近零”的曝光时间），以生成示出的细胞轧制6,7的一步一步的进展的图像序列。双摄像头发射分光系统能满足这些要求的成像单元带有多种荧光团。 双摄像发射分光系统拆分和滤波器荧光通道分成两个相似的摄像机同时捕获两个空间上相同，但荧光团特定的图象，同时保留的全视场。该技术使实时在每个通道中捕获的图像进行直接比较，并允许用户快速照相机模型之间具有不同的成像能力切换。此功能可用于制作在一个摄像头，更好地让系统捕捉调整图像捕捉设置有用荧光基团具有不同强度，寿命和消光系数8。再加上成像软件，双摄像头发射分光系统允许在多个波长活细胞成像分析的实时记录，并可以提高在体外实验使用的荧光来研究细胞行为。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>BT-20 cells</td>
 <td>ATCC</td>
 <td>HTB-19</td>
 <td></td>
 </tr>
 <tr>
 <td>CHO-E cells</td>
 <td>Gift from Dr. R. Sackstein (Brigham and Women's Hospital, Harvard Medical School)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>MEM </td>
 <td>Thermo Scientific</td>
 <td>SH30024.01</td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Thermo Scientific</td>
 <td>SH30396.03</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>Thermo Scientific</td>
 <td>SV30010</td>
 <td></td>
 </tr>
 <tr>
 <td>0.25% Trypsin / 0.1% EDTA</td>
 <td>Thermo Scientific</td>
 <td>SV30031.01</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Thermo Scientific </td>
 <td>SH30028.02</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS+</td>
 <td>Life Technologies</td>
 <td>14080-055</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A9647</td>
 <td></td>
 </tr>
 <tr>
 <td>HECA-452 monoclonal antibody</td>
 <td>BD Biosciences</td>
 <td>555946</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-human CD24 monoclonal antibody</td>
 <td>BD Biosciences</td>
 <td>555426</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-rat IgM AlexaFluor 488</td>
 <td>Invitrogen</td>
 <td>A21212</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse IgG AlexaFluor 568</td>
 <td>Invitrogen</td>
 <td>A11004</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>[header]</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>EXi Blue Fluorescence Microscopy Digital CCD Camera</td>
 <td>Q Imaging</td>
 <td>EXI-BLU-R-F-M-14-C</td>
 <td></td>
 </tr>
 <tr>
 <td>Retiga EXi FAST 1394 Digital CCD Camera</td>
 <td>Q Imaging</td>
 <td>RET-EXi-F-M-12-C</td>
 <td></td>
 </tr>
 <tr>
 <td>DC2 Emission Splitter</td>
 <td>Photometrics</td>
 <td>DC2 </td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted Fluorescence Microscope</td>
 <td>Leica</td>
 <td> DMI6000 B</td>
 <td></td>
 </tr>
 <tr>
 <td>Streampix 5 software</td>
 <td>Norpix</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

simultaneously capturing real-time images in two emission channels using a dual camera emission splitting system: applications to cell adhesion

多色免疫荧光显微镜来检测特定的分子在细胞膜上可以加上平行平板流动腔实验来调查管动态流动条件下的细胞粘附机制。例如，标有多种荧光团的癌细胞可灌注了一个潜在的反应底物来建模癌转移的机制。然而，多通道单相机系统和彩色摄像机表现出的缺点在图像采集的实时活细胞分析。为了克服这些局限性，我们采用了双摄像头发射分光系统同时捕获荧光标记细胞的实时图像序列中的流室。双摄像头发射分光系统过滤器中定义的波长范围分为两个黑白CCD摄像机，从而同时捕获两个空间相同，但荧光的特定图像。随后，psuedocolored 1通道的图像被组合成一个单一的实时合并序列，可以揭示多个目标分子对细胞运动迅速在一个地区的利益。

平行板流动腔粘连实验检测证明，同时捕获实时图像序列的两个发射信道的双摄像头发射分光系统（ 图1）。双相机发射分光系统检测出BT-20细胞，进行了荧光标记的抗人CD24和HECA-452（检测sialofucosylated抗原）的单克隆抗体和合适的第二抗体（ 图2）。一些细胞滚动显示红色信号（CD24）还检测不到绿色信号（HECA-452），其他显示绿色信号和检测不到的红色信号，并且还在其?...

Watch this Scientific Journal Video about Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion at JoVE.com

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

双摄像头发射分光系统，双色荧光显微镜生成具有出色的光学分辨率和时间分辨率，一定的活细胞检测的要求，包括平行板流动腔粘连检测的实时图像序列。当软件被用来从合并收购的同时发射通道的图像，伪彩色图像序列产生。

同时捕获实时图像在两个发射通道采用了双摄像头发射分光系统：应用到细胞粘附

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

simultaneously-capturing-real-time-images-two-emission-channels-using

Research

JoVE Journal

Bioengineering

9.5K 次观看.  Ohio University. 双摄像头发射分光系统，双色荧光显微镜生成具有出色的光学分辨率和时间分辨率，一定的活细胞检测的要求，包括平行板流动腔粘连检测的实时图像序列。当软件被用来从合并收购的同时发射通道的图像，伪彩色图像序列产生。 

视频: Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

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

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

研究微加工通道细胞迁移的

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

在细胞和组织力学生物学应用一种新型的拉伸平台

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

手持式实时非侵入性小动物成像的临床光声成像系统

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved oxygen concentration). Nevertheless, the morphology of cells can be a suitable indicator for optimal conditions, since it changes with dependence on the growth state, product accumulation and cell stress. Furthermore, the single-cell size distribution provides not only information about the cultivation conditions, but also about the population heterogeneity. To gain such information, a photo-optical in situ microscopy device1 was developed to enable the monitoring of the single-cell size distribution directly in the cell suspension in bioreactors. An automated image analysis is coupled to the microscopy based on a neural network model, which is trained with user-annotated images. Several parameters, which are gained from the captures of the microscope, are correlated to process relevant features of the cells, like their metabolic activity. Until now, the presented in situ microscopy probe series was applied to measure the pellet size in filamentous fungi suspensions. It was used to distinguish the single-cell size in microalgae cultivation and relate it to lipid accumulation. The shape of cellular particles was related to budding in yeast cultures. The microscopy analysis can be generally split into three steps: (i) image acquisition, (ii) particle identification, and (iii) data analysis, respectively. All steps have to be adapted to the organism, and therefore specific annotated information is required in order to achieve reliable results. The ability to monitor changes in cell morphology directly in line or on line (in a by-pass) enables real-time values for monitoring and control, in process development as well as in production scale. If the off line data correlates with the real-time data, the current tedious off line measurements with unknown influences on the cell size become needless.

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

在原位生物过程单细胞形态实时测定的显微镜

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

在微生物发酵过程中制造一种纳菲安涂层、还原石墨烯 Oxide/Polyaniline 化学电阻传感器, 以实时监测 ph 值

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...