Name: acquiring fluorescence time-lapse movies of budding yeast and analyzing single-cell dynamics using grafts
Uploaded: 2013-07-18T13:00:00
Description: Watch this Scientific Journal Video about Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS at JoVE.com

我们感谢刘慧卿杰克逊，约书亚蔡德曼，和尼古拉斯·雷恩对软件的意见。这项工作是由GM95733（NM），BBBE 103316和麻省理工学院的启动资金（NM）。

1。获取单个酵母细胞生长在微流控室的荧光显微镜电影<ol><li>培养物接种1毫升SC媒体（合成用2％的葡萄糖和氨基酸的完整补充定义的介质）与细胞从新鲜生长板，和孵育在30℃下培养过夜〜16小时的辊鼓上准备培养，例如，最终的OD 600nm的 〜0.1早期的对数相生长。 </li><li> OD 600nm的 = 0.1稀释起动文化需要，新生1毫升新鲜试管6-8小时。这提供了适合微流体装置装入密度细胞生长在?...

<ol>
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上述协议描述了一个简单的方法来获取和分析经验有限，在微流或在软件开发的的荧光时间序列数据。它允许一个取得时移荧光电影单个酵母细胞中提取相关的细胞大小和表达测量;牧师跟踪和血统分配和整个细胞的行为进行分析，随着时间的推移，使用市售的微流体培养装置和一个多功能的图形用户界面（GUI）。实验，分割和跟踪步骤已经以各种方式以前11,13,14接近，上面的程序的目的?...

单细胞基因表达分析进一步了解基因调控的许多方面。荧光记者表达的静态快照，使用流式细胞仪或显微镜单细胞表达的分布提供了有用的信息，但缺乏需要直接告知基因表达的动态时间序列数据的历史和演变。荧光时间推移显微镜呈现的一种手段，同时获得单细胞测定和他们的历史。各种实验和分析技术已经发展到获取和定量荧光记者表达的电影，从而传授洞察（见检讨1）如细胞与细胞变异细菌的持久化形成4，2,3，转录基因调控功能启动和延伸5，转录6,7爆破，细胞周期依赖性8,9的 ，和遗传10。但是，OBT质量恒常单细胞荧光时间序列，包括培养的单层细胞在一个可控的环境中，并在高通量定量分析所获取的荧光电影的重要的技术挑战。在这里，我们描述一个过程来获取和分析荧光S的电影酵母中没有所需的经验，在细胞培养装置制造或在软件开发中（ 图1）。 首先，我们将详细地，例如，协议产生荧光的时间序列的芽殖酵母表达一种或多种荧光记者的电影。虽然定制的微文化室已建成并成功地采用了先前11-13，我们使用市售的微流体装置从CellAsic（加利福尼亚Hayward）。系统局限单层生长的细胞，并允许灌注环境的持续控制。显微镜协议，我们提出的是一个简单的方法来获得fluoresc芽殖酵母的ENCE电影的，但任何修改过的实验方案（定制培养装置，替代介质条件等 ）的单个酵母细胞产生类似的荧光电影数据可能会被取代。 接下来，我们勾勒出电影使用的图形用户界面（GUI）基于MATLAB软件包（Mathworks公司，马萨诸塞Natic​​k），被称为快速荧光时间​​系列分析的图形用户界面（移植物），提取时间序列数据分析单细胞。移植物有类似的功能，多功能的，开放源码的软件包细胞ID 14分割和跟踪细胞中提取荧光强度和几何信息。然而，移植物提供了重要的额外功能。首先，它提供了简单的交互式编辑分割和跟踪结果来验证数据的准确性，而不是仅仅统计浇注离群区域分析后的痕迹。此外，它扩展了分析，自动生成Ÿ候谱系和细胞周期的芽殖酵母的兴趣点。决定时，母亲和女儿划分，以形成两个独立的单元区域是非常重要的，以确定在整个细胞周期8的全细胞（包括任何连接芽的母亲）测量。该套件包括三个模块来完成这些任务。第一部分单元区域的基础上聚焦和散的明视场图像之间的对比度，并允许用户定义和视觉测试分段参数。第二轨道（布莱尔和克罗克等 IDL例行杜弗兰的MATLAB实现，可在： <a href="http://physics.georgetown.edu/matlab/" target="_blank">http://physics.georgetown.edu/MATLAB/</a> ），通过测量细胞区域自动分配谱系;使视力检查和纠错。一个简单的绘图GUI是来查询单细胞性质。第三个模块赋予现蕾和分工TIMES，和全细胞输出的时间序列数据，以及他们的第一和第二时间的衍生物（9所讨论的）。分析模块输出的数据，为后续研究的统计软件选择作为空间分隔的文本文件。因此，包使用户能够提取高品质的时间序列数据，通过图形界面。我们已经用这种方法在单芽殖酵母细胞的细胞周期9为一个函数估计实时转录率。模块进行了优化，芽殖酵母，参数或，如有必要，可适于其他生物体和图像类型可自由查看的代码。分割，跟踪和谱系分配算法可能是特定类型的成像分配和生物体。现有的算法可以被取代，但仍保留的GUI界面，允许用户友好的可视化分割和跟踪检查和纠正错误总是占据R与任何算法。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Y04C Yeast Perfusion Plate</td>
			<td>CellAsic</td>
			<td>Y04C-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ONIX Microfluidic Perfusion Platform</td>
			<td>CellAsic</td>
			<td>EV-262</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Axio Observer.Z1 Microscope</td>
			<td>Zeiss</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plan-Apochromat 63X/1.40 Oil DIC objective</td>
			<td>Zeiss</td>
			<td>440762-9904-000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cascade II EMCCD camera</td>
			<td>Photometrics</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lumen 200 metal-halide arc lamp</td>
			<td>PRIOR Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triple-bandpass dichroic filter cube and excitation and emission filter set</td>
			<td>Chroma Technology Corp</td>
			<td>set #89006</td>
			<td>Used for YFP (Venus/Citrine), CFP (Cerulean), RFP (mCherry/tdTomato)</td>
		</tr>
		<tr>
			<td>MAC 5000 controller and filter wheels</td>
			<td>Ludl Electronic Products</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MATLAB R2011a</td>
			<td>Mathworks</td>
			<td>&nbsp;</td>
			<td>64-bit version handles large data files better than 32-bit</td>
		</tr>
	</tbody>
</table>

acquiring fluorescence time-lapse movies of budding yeast and analyzing single-cell dynamics using grafts

荧光时间推移显微镜在单细胞水平研究的许多生物过程中已经成为一个强大的工具。特别地，描绘基因表达的时间依赖性的电影提供深入了解其调节的动态，但是，也有许多的技术挑战，以获得和分析单个细胞的荧光电影。我们在这里介绍一个简单的协议，使用市售的微流体培养装置，产生这样的数据，和一个基于MATLAB的图形用户界面（GUI）为基础的软件封装量化荧光图像。的软件段和轨道细胞，使用户能够直观地牧师数据中的错误，并自动分配的血统和分裂次数。 GUI进一步分析时间序列产生整个细胞的痕迹以及他们的第一和第二次衍生物。虽然该软件是专为S。酵母 ，其模块化和通用性，应该允许它吨Ø作为一个平台，研究其他细胞类型的一些修改。

成功完成和分析的实验将产生大多是连续的时间序列与实际分配的芽和分裂次数的单全细胞。作为一个例子，我们履行上述协议ADH1启动子组成，观察增长和全球表现可能会有所不同随着时间的推移在单细胞（ 表4，Y47带动表达天蓝荧光蛋白（CFP）的整合拷贝单倍体酵母菌株）。我们跑了时间序列分析，获得整个细胞（ 图7A）测量随着时间的推移，9音...

Watch this Scientific Journal Video about Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS at JoVE.com

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

我们提出了一个简单的协议来获得生长的酵母细胞的荧光显微镜的电影，和一个基于GUI的软件程序包，提取单细胞的时间序列数据。该分析包括自动沿袭和集成的跟踪数据的目视检查和手工curation分组表决的时间分配。

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Research

JoVE Journal

Biology

Measuring Replicative Life Span in the Budding Yeast

在芽殖酵母中的自我复制寿命测量

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

蛋白质纯化技术，可以检测SUMO化修饰和芽殖酵母蛋白动粒和Ndc10泛素Ndc80的

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

高分辨率成像和芽殖酵母个人星体微管动力学分析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...