我们感谢生物成像技术发展中心（BIDC）美国加州大学，旧金山Imaris，Imaris XT和Matlab使用。我们提供的技术援助和亨利，LK弗洛伦所作出的贡献的手稿的编辑，L. Daitch感谢Kharazia五。这项工作是由从加利福尼亚州医学研究的加州大学旧金山分校的酒精和药物滥用通过SEB，美国国立卫生研究院的资助：1R21DA029966-01和美国国立卫生研究院快速轨道奖励，到筛选的MLSMR，收集到SEB，加州大学旧金山分校药学院（院长办公室和临床药学）和医学院（临床药理学和实验疗法）CLHK。

1。三维形态分析单细胞的表型变化<ol><li>转染的人类胚胎肾（HEK293）细胞与血凝素（HA）的标签的促肾上腺皮质激素释放因子受体-2（CRF-R 2），G蛋白偶联受体（GPCR），前面描述的4，5。 </li><li>被留下细胞未经处理的（无处理，NT），CRF-R2的内源性配体，促肾上腺皮质激素释放因子，慢性肾功能衰竭（1μM，30分钟），或预处理CRF-R2的选择性拮抗剂，抗-SAUVAGINE 30（AS刺激-30，1μM，30分钟）激动剂治疗之前。 </li><li>然后将细胞固定，透化和用抗-HA。 CRF-R2采用Alexa的594 nm的共轭反鼠抗体（IgG抗体）。 DAPI用于可视化的细胞核的有丝分裂阶段。 </li><li>要限制实验者主观性，直到后的图像进行采集和分析的实验条件下，不知道。 </li><li>我们获取的图像从固定的HEK293细胞使用计划“复消色差透镜63x/1.4油DIC的目标和连接到相干集成双光子激光系统包括一个威尔第-V5激光和米拉900-F激光系统蔡司LSM 510 META共聚焦显微镜。 </li><li>在数据采集过程中，将细胞条块由多光谱切片，488纳米和790纳米（〜350 nm的2PH实施例）和z-分区（0.5μm的增量），以包括数据从核膜的外细胞外受体四肢。 </li><li>的荧光数据的第一被处理使用Imaris，它允许三维显微镜的可视化和分割数据集，和立方体素组成一个三维模型创建的形态学分析。 然后，Imaris XT模块接口Imaris利用MATLAB计算机程序语言，以确定该点的坐标的G蛋白偶联受体扩展。 要考虑到帐户细胞变异，我们得到的荧光图像分析德EN 22细胞：没有治疗（NT）组（n = 7），激动剂（CRF）治疗组（n = 8）和预处理拮抗剂（AS-30）之前激动剂治疗组（n = 7）（ 图2） 。 <ol><li>感兴趣区域（ROI）应该包括一个电池，它是不活跃的有丝分裂阶段，而不是到其他细胞。以这种方式，分析将包括只有一个核和受体扩展的细胞与未扰动由相邻的其他细胞。 </li><li>细胞的三维结构重建多光谱荧光数据，用Imaris（v.7.1.1）。 </li><li>继Imaris设计的算法，第一表面渲染被用来代表核膜。 imaris将确定是否有多于一种的原子核中的ROI。 </li><li>然后点创建算法，利用定位CRF-R2扩展的。利用点检测，因为它可以补偿背景噪声和不规则的强度非晶形细胞的复杂网络。 </li><li>为了最大限度地提高列入CRF-R 2的每个单元的荧光检测，斑点的直径被设定为0.2微米，这是最小的单元，在图像内的不同的信息的形式使用的高斯滤波器的测量强度推断。现货过滤成立于点自动创建过程。 ，但是，该软件使用户可以灵活使用过滤器来定义的参数。 </li><li>为了避免数据被截断，数据集被转换8位（无符号）固定点，32位小数。 </li><li>体素的强度数据被交换，以发现使用Imaris XT模块用MATLAB接口和每个点的确切的空间位置的坐标数据，被确定作为一个参考点（ 图3）执行的距离变换使用核膜。 </li></ol></li><li>由此产生的数据可以量化，并以图形格式为statistical分析。组间比较采用双向方差分析和Bonferroni后测。数据以平均值±标准差。 * P &lt;0.05，差异被认为是显着的。 GraphPad棱镜5.02（ 图4）进行了计算。 </li></ol> 2。代表性的成果为了证明我们的方法的功率，我们量化的细胞变化，从G蛋白偶联型受体（GPCR），促肾上腺皮质激素释放因子受体2（CRF-R 2），而其内源性配体在转染的HEK293细胞的慢性肾功能衰竭的相互作用的结果。 我们表明，CRF-R2受体位于从有限区域的膜的细胞（ 图2A和电影1）在质膜和项目。使用传统的二维分析，也能够检测到这个子集外的CRF-R2受体只有当我们分析受体粘附婆int型的玻璃覆盖。因此，我们失去任何其他的信息来自Z-堆叠的多光谱数据（ 图5）。 治疗慢性肾功能衰竭，当细胞的胞外受体被大大降低，如所示的斑点从质膜的距离的减少。他们也从主要有限的位置重新分配到一个数量的离散的位置（ 图2B和电影2）。 CRF受体膜分布的影响，防止预处理与CRF-R2的特定受体拮抗剂，antisavagine 30（AS-30），我们发现，CRF-R2扩展的不改变（ 图2C和电影3）。 斑点的前端分布，绘制在5微米的频谱颜色编码的时间间隔，利用可视化的体素之间的距离从核膜。无治疗和拮抗剂pretreatmENT（AS-30，1μM，30分钟）前（CRF受体激动剂治疗，1μM，30分钟），差异没有显着性差异（NS）在G蛋白偶联受体收缩。逐步激动剂（CRF，1μM，30分钟）处理的细胞与CRF-R 2含的体素的数量减少时相比，没有治疗，为6-15微米（0-5微米（纳秒），** P &lt; 0.01）和&gt;15μm的（*** P &lt;0.005），或相比，AS-30的治疗，为11-15微米（0-10微米（纳秒），** P &lt;0.01）和&gt;15μm的（*** P &lt;0.005）（ 图4）。 <img alt="图1" src="/files/ftp_upload/4233/4233fig1.jpg" /> 图1。目前可用的技术和荧光图像分析的限制。图<a href="http://www.jove.com/files/ftp_upload/4233/4233fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/4233/4233fig2.jpg" /> <strong图2转染的HEK293细胞的HA-CRF-R2激动剂和拮抗剂治疗前后的三维多光谱荧光图像。合并后的影像显示HA-CRF-R2探测用抗-HA和可视化使用Alexa 488标记的抗鼠球蛋白（IgG）二次抗体，DAPI是用于可视化的核。激光共聚焦扫描显微镜（CLS）的图像被收购。比例尺为5μm。 <img alt="图3" src="/files/ftp_upload/4233/4233fig3.jpg" /> 图3 HA-CRF-R2 HEK293转染细胞的三维模型重建CLS使用Imaris软件。表面绘制的核心和描述转换成小泡的G蛋白偶联受体的扩展点创建。荧光数据处理的Imaris这使得3D显微镜数据集的可视化和细分化。然后，利用Imaris XT与MATLAB接口Imaris。兑换成体素强度点的坐标。斑点的频谱的编码的颜色（蓝色0-5微米，绿色，6-10微米，黄色11-15微米和红色&gt; 15微米）代表的距离表格核膜。规模500微米。 <img alt="图4" src="/files/ftp_upload/4233/4233fig4.jpg" /> 图4绘制的图形表示的前端的斑点分布在5微米的间隔的频谱颜色编码是利用从核膜的体素进行可视化的距离。拮抗剂（AS-30，1μM，30分钟）（CRF受体激动剂治疗前，1μM，30分钟）没有处理和预处理差异没有显着性差异（NS）在G蛋白偶联受体收缩。激动剂（CRF，1μM，30分钟）处理的细胞与逐步降低CRF-R 2含的体素的数目相比没有治疗0-5微米（ns）， ​​其中6-15微米（** P的距离&lt;0.01），&gt; 15微米（*** P &lt;0.005）;或AS-30治疗0-10微米（NS），11-15<html>微米（** P &lt;0.01）和15微米（*** P &lt;0.005）。 <img alt="图5" src="/files/ftp_upload/4233/4233fig5.jpg" /> 图5。HEK 293转染细胞的HA-CRF-R2的的二维形态分析的限制。中平面部分的细胞（3-4μm的上方的玻璃盖玻片），示出了中心的细胞核用DAPI和HA-CRF-R2可视化探测使用抗-HA和可视化使用Alexa的488nm波长的共轭抗小鼠（IgG抗体1）二次抗体和收购CLS（A）不治疗（NT）和（B）的激动剂（慢性肾功能衰竭，1μM，30分钟）没有什么区别，而的受体粘合点是显着不同的。 动画1。自由旋转的3D模型中的“超越”模式的HEK 293 HA-CRF-R2，没有治疗，转染细胞受体蛋白的细胞表型之间的差异进行评估。为5〜20微米的比例尺。/ files/ftp_upload/4233/4233movie1.avi“目标=”_blank“&gt;点击这里观看电影。 电影2，可自由旋转的3D模型中的“超越”模式的HEK 293转染的细胞与HA-CRF-R2，激动剂治疗，慢性肾功能衰竭（CRF，1μM，30分钟），以评估受体蛋白质的细胞表型差异。比例尺为5至20微米。 <a href="http://www.jove.com/files/ftp_upload/4233/4233movie2.avi" target="_blank">点击这里观看电影。</a> 电影3自由旋转的3D中的“超越”模式的HEK 293转染的细胞与HA-CRF-R2，激动剂治疗的拮抗剂（AS-30，1μM，30分钟）前的预处理（CRF，1μM，30分钟），以评估受体蛋白质的细胞表型差异。比例尺为5至20微米。 <a href="http://www.jove.com/files/ftp_upload/4233/4233movie3.avi" target="_blank">点击这里观看电影。</a>

<ol>
	<li>West, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design-based+stereological+methods+for+counting+neurons.">Design-based stereological methods for counting neurons.</a> Prog, Brain Res. 135, 43-51 (2002).</li><li>Burke, M., Zangenehpour, S., Mouton, P. R., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knowing+what+counts:+unbiased+stereology+in+the+non-human+primate+brain.">Knowing what counts: unbiased stereology in the non-human primate brain.</a> J. Vis. Exp. (27), e1262 (2009).</li><li>Sugawara, Y., Ando, R., Kamioka, H., Ishihara, Y., Honjo, T., Kawanabe, N., Kurosaka, H., Takano-Yamamoto, T., Yamashiro, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+three-dimensional+morphometry+and+cell-cell+communication+of+the+osteocyte+network+in+chick+and+mouse+embryonic+calvaria.">The three-dimensional morphometry and cell-cell communication of the osteocyte network in chick and mouse embryonic calvaria.</a> Calcif. Tissue Int. 88, 416-424 (2011).</li><li>Vickery, R. G., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+dynamin-dependent+and+-independent+mechanisms+target+structurally+homologous+dopamine+receptors+to+different+endocytic+membranes.">Distinct dynamin-dependent and -independent mechanisms target structurally homologous dopamine receptors to different endocytic membranes.</a> J. Cell Biol. 144, 31-43 (1999).</li><li>Bartlett, S. E., Enquist, J., Hopf, F. W., Lee, J. H., Gladher, F., Kharazia, V., Waldhoer, M., Mailliard, W. S., Armstrong, R., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+responsiveness+is+regulated+by+targeted+sorting+of+D2+receptors.">Dopamine responsiveness is regulated by targeted sorting of D2 receptors.</a> Pro.c Natl. Acad. Sci. U.S.A. 102, 11521-11526 (2005).</li><li>Gordon, A., Colman-Lerner, A., Chin, T. E., Benjamin, K. R., Yu, R. C., Brent, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+quantification+of+molecules+and+rates+using+open-source+microscope-based+cytometry.">Single-cell quantification of molecules and rates using open-source microscope-based cytometry.</a> Nat. Methods. 4, 175-181 (2007).</li><li>Schock, F., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+epithelial+morphogenesis.">Molecular mechanisms of epithelial morphogenesis.</a> Annu. Rev. Cell Dev. Biol. 18, 463-493 (2002).</li><li>Pincus, Z., Theriot, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+quantitative+methods+for+cell-shape+analysis.">Comparison of quantitative methods for cell-shape analysis.</a> J. Microsc. 227, 140-156 (2007).</li><li>Spiller, D. G., Wood, C. D., Rand, D. A., White, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+single-cell+dynamics.">Measurement of single-cell dynamics.</a> Nature. 465, 736-745 (2010).</li><li>Loo, L. H., Wu, L. F., Altschuler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+multivariate+profiling+of+drug+responses+from+single+cells.">Image-based multivariate profiling of drug responses from single cells.</a> Nat Methods. 4, 445-453 (2007).</li><li>Yarrow, J. C., Totsukawa, G., Charras, G. T., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+cell+migration+inhibitors+via+automated+microscopy+reveals+a+Rho-kinase+inhibitor.">Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor.</a> Chem. Biol. 12, 385-395 (2005).</li></ol>

没有利益冲突的声明。

我们发现，慢性肾功能衰竭治疗引起的形态和位置的CRF-R2的一个显着的变化。被抑制的选择性拮抗剂治疗CRF-R2的变化。我们表明，受体修改未检测到不能被测量，使用标准的2D多光谱技术。将复杂的生物参数形态分析研究复杂3D图像的能力是至关重要的。我们没有预先定义的形状不一致的荧光网络组件，能够使细胞的三维测量。我们的套件，用户可修改的自动化的方法，使我们能够量化的分子动力...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>人类胚胎肾（HEK293） </td><td>美国典型培养物保藏中心</td><td> CRL-1573 </td><td></td></tr><tr><td> Dulbecco改良的Eagle培养基（DMEM） </td><td> Invitrogen公司</td><td> 11965118 </td><td></td></tr><tr><td>胎牛血清（FBS） </td><td> Invitrogen公司</td><td> SH30070.03 </td><td></td></tr><tr><td> AlexaFluor-488（IgG2b亚） </td><td> Invitrogen公司</td><td> A-11001 </td><td></td></tr><tr><td>单克隆抗-HA.11的（IgG抗体1） </td><td>科文斯</td><td> 16B12 </td><td></td></tr><tr><td> DAPI </td><td>矢量实验室</td><td> H-1200 </td><td>; </td></tr><tr><td> CRF </td><td>西格玛</td><td> C2917 </td><td></td></tr><tr><td> antisauvagine-30（AS-30） </td><td>西格玛</td><td> A4727 </td><td></td></tr></tbody></table>

an analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images

最常见的软件分析工具，可用于测量荧光图像的二维（2D）数据的纳入和排除的数据点，和计算机辅助模式识别的分析结果的解释和支持，依靠手动设置。它已成为日益重要的是能够测量从三维（3D）数据集构成的荧光图像，以便能够捕捉的复杂性的细胞动力学和理解的基础上的细胞在生物系统内的可塑性。多光谱荧光图像和强大的分析软件重建的共聚焦图像的堆栈，然后收集到的二维图像的三维表示，通过收购，允许复杂的显微镜工具的可视化三维荧光图像。先进的设计为基础的体视学方法已经由近似和假设的origina基于L型体视学1，即使在复杂的组织切片2。在显微镜尽管有这些科学的进步，仍需要一个自动化的分析方法，充分利用了固有的三维数据，以便分析和定量分析的复杂变化细胞形态，蛋白定位和受体贩运。 目前的技术可量化的荧光图像变形元（分子器件，桑尼维尔，CA）和图像J（NIH）提供人工分析。 Imaris（安道尔技术，贝尔法斯特，北爱尔兰）软件提供了的功能MeasurementPro，可以手动创建的测量点可以被放置在一个体积图像或一系列二维切片创建一个三维对象上绘制。此单击单点测量，测量的线的两个对象之间的距离，或创建一个多边形包围的感兴趣区域的方法是有用的，但它是难以适用于并发症前蜂窝网络结构。灯丝示踪（安道尔）允许自动长丝般然而，此模块已经发展到测量定义的结构，如神经元，该神经元树突，轴突和棘（类似树的结构）是由神经元的三维检测。此模块已巧妙地利用，使形态测量非神经元细胞3，然而，输出数据的一个扩展的蜂窝网络提供的信息，通过使用定义的细胞的形状，而不是作为非晶形的细胞模型依赖于一个软件，。为了克服这个问题的分析非晶形细胞，使软件更适合的生物应用，，Imaris开发Imaris细胞。这是一个科学项目与EidgenössischeTECHNISCHE Hochschule的，已经发展到细胞和细胞之间的关系计算。虽然该软件可以检测的生物制约，迫使每一个核细胞使用到段细胞的细胞膜上，它可以不被用来分析的荧光数据是不连续的，因为理想地建立细胞表面无空隙空间。据我们所知，目前还没有用户可修改的形态测量信息的自动化解决方案，可提供三维荧光图像已被开发，可实现细胞的空间信息未定义的形状（ 图1）。 我们已经开发了一个分析平台采用的Imaris核心软件模块和Imaris XT连接到MATLAB（垫厂，公司）。这些工具允许细胞的三维测量的，没有一个预定义的形状，和不一致的荧光的网络组件。此外，这种方法将允许研究人员已扩大在生物系统的专业知识，但不熟悉计算机应用，细胞动力学的形态变化进行量化。

Watch this Scientific Journal Video about An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images at JoVE.com

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

我们开发了一个软件平台，利用神经科学，ImarisXT和MATLAB Imaris未定义形状的三维共聚焦荧光的单细胞形态的变化来衡量。这种新方法可以用来量化受体激活后，细胞形状的变化，因此，为药物发现代表一个可能的额外的工具。

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

an-analytical-tool-that-quantifies-cellular-morphology-changes-from

Research

JoVE Journal

Biology

14.5K 次观看.  University of California, San Francisco. 我们开发了一个软件平台，利用神经科学，ImarisXT和MATLAB Imaris未定义形状的三维共聚焦荧光的单细胞形态的变化来衡量。这种新方法可以用来量化受体激活后，细胞形状的变化，因此，为药物发现代表一个可能的额外的工具。

视频: An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

基于芯片的三维细胞培养，在灌注微生物反应器

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

科研

生物学

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

变化在大鼠乳腺形态和患乳腺癌的风险

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...