Name: an analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images
Uploaded: 2012-08-31T13:00:00
Description: Watch this Scientific Journal Video about An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images at JoVE.com

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）?...

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	<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>

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 ...

Research

JoVE Journal

Biology

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

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

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

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

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

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

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

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

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 ...

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

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

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 ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

单个细胞分辨率三维成像的有机体

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...