作者要感谢阿曼达Struckhoff与实验的初始帮助。这项工作是从5RO1CA115706 NIH的拨款支持。

1。胶原涂层板的制备<ol><li>胶原蛋白稀释到终浓度为10微克/毫升的Opti-MEM媒体。 </li><li>大衣胶原的6孔板，从第1步，每孔加入1.5毫升。孵育过夜在4°C </li><li>翌日，1X磷酸的2倍缓冲液（PBS）洗板，将它们存储在PBS。 </li></ol> 2。在6孔板细胞的制备及幼苗 注:用温水媒体和PBS，以确保细胞运动的最佳条件<ol><li>成长的MDA-MB-231（浸润性乳腺癌细胞株）在杜尔贝科的改良Eagle培养基（DMEM培养基），10％胎牛血清（FBS），人口稀少。在这个例子中，我们使用的MDA-MB-231，然而，这种方法可以应用到任何附着的癌变和非癌变细胞株。 </li><li>温暖1X PBS洗涤细胞两次和收集由胰蛋白酶。检查细胞在显微镜下，使苏重新正确胰蛋白酶处理细胞分离。 </li><li>收集细胞，加入含10％胎牛血清的DMEM培养基，轻轻地旋转5分钟，在1000Ğ细胞。重悬于含10％胎牛血清的DMEM培养基中的细胞沉淀轻轻。 </li><li>移液器上下轻轻打破细胞沉淀。重要的是尽可能地分解成单个细胞的细胞团。 </li><li>计数细胞，并与10％胎牛血清的DMEM培养基50,000个细胞/毫升的终浓度稀释。免除2毫升到每口井。 </li><li>板在37°C，在组织文化的孵化器孵育过夜，直到出发的时间推移显微镜。 </li><li>迁移，以确保足够的空间，创建一个伤口，用枪头，用1X PBS洗板清除杂物（如伤口形成），并加入含10％胎牛血清的DMEM培养液。检查显微镜下的浮动细胞。如果大量的细胞是浮动的，重复洗涤过程。现在，该板块是准备上的设置显微镜图像采集。 </li></ol> 3。显微镜的安装和自动图像采集有几个控制自动图像采集显微镜的成像软件包。在这里，我们使用Slidebook 5.0图像处理软件和1奥林巴斯显微镜（奥林巴斯IX81）耦合到EM-滨松C9100相机，这是极好的活细胞应用是最重要的收购速度和最小的光毒性，进行自动采集。该系统包括一个舞台上方的环境控制腔（NEUE活细胞）和自动化的XY平台控制器（之前Proscan的）。一个10X UPLANFL Ph1/0.30目标，在明亮的场模式使用规定的时间间隔，图像采集。以下协议描述的图像采集。 <ol><li>打开显微镜，照相机，和活细胞成像设备。定位板在微观阶段。覆盖板的"新活细胞ENVIronmental控室，这是一个CO 2供应和温度传感器连接。温控器设置在37°C和5％的CO 2。 </li><li> SlideBook软件可以完全自动化的目标选择，波长选择和荧光和亮场技术之间轻松切换。为了设置不同的参数控制的重点，开放SlideBook软件&gt;去菜单栏&gt;选择重点控制，通过点击&gt; <img alt="图0按钮1" src="/files/ftp_upload/3585/3585fig0btn1.jpg" />并选择以下参数: <ol><li>在"目标"框中，"确定从下拉窗口下拉选择的目标。请注意，我们使用了一个阶段的目标和冷凝器手动选择适当的阶段环。如果使用了自动冷凝器系统，这将自动设置通过软件程序。另外，如果既不客观，炮塔也冷凝器自动化这些应该每个人都可以选择手动LY。 </li><li>在"过滤设置"框: <ol><li>选择"设置"直接光路目镜或相机从下拉框可用的过滤器（"活"的目镜，"广角"相机在我们的系统）。 </li><li>明照明选择适当的过滤器（在我们的设置，此选项被标记为"DIC的"明光路为代表的开放位置）。 </li><li>选择"开亮"点击打开明亮的领域快门的光源。 </li></ol></li></ol></li><li>要查看下眼片样品，选择明亮的领域炮塔上的过滤器（在我们的系统中的位置"6"）。在最初通过目镜观察样品，找到合适的领域，并带来成焦点（步骤3.2.1 3.2.2.3）的样品。将过滤器的炮塔改变光路图像采集（位置"1"在我们的系统）的相机。 </li><li>自动化阶段允许选择不同的XY coordina附加费捕捉图像采集过程中利益的多个领域。在SlideBook软件，到重点控制，选择"XY"，选择不同的岗位虽然眼睛片，然后按一下"设定点"锁定在每个位置图像采集（ 图1）。 </li><li>微调的摄像机图像，在屏幕上观看的焦点。这可以通过使用手动控制，在显微镜或电脑控制，可在更精细的增量调整的重点。 </li><li> "焦点控制"窗口中使用的控制，调整z位置的阶段。为达到最佳效果，重点对附近的板接触的扁平细胞突起。 </li><li>为了帮助重点使用滑块来调整曝光时间，在适当的范围内为重点。如果多个XY位置已选定，为每个位置调整的重点:重点控制&gt;的XY&gt;访问点下。注:这样做是为了确保，日领域是正确的集中最优的快照。 </li><li>使用该软件来设置捕获窗口的参数。在SlideBook，打开所选择的捕捉<img alt="图0按钮2" src="/files/ftp_upload/3585/3585fig0btn2.jpg" />从菜单栏。 见图2。然后，我们检查以下参数。 <ol><li>检查捕获类型&gt;随着时间的推移。 </li><li>检查过滤器设置:大视野:迪爱生（亮场图像）。 </li><li> "时间流逝捕获"是时间点的数量，将捕获的（它可以有所不同，取决于细胞类型）。时间是实验的时间总长度。从下拉菜单中可以选择的时间单位毫秒（ms），秒（s），分钟（m）或小时（h）] [。 </li><li> "间隔"是延迟之间的一个时间点开始，在接下来的时间点开始。当你输入两个值，第三个字段将自动计算。 </li><li>多个XY采集:选择"多点名单"超过1 XY位置。 </li><li>命名该文件。在给定的例子，我们把它命名为&#39;231&#39;。 </li></ol></li><li>为了避免意外关闭计算机由于多个图像，使用"假脱机选项"保存文件。它是可选的，但它是强烈建议。为此，捕捉下contro升&gt; 选择高级 &gt; 阀芯 &gt; 捕获到内存中，并保存后各时间点假脱机文件。浏览的位置来保存幻灯片书文件（ 图3）。后来从指定的位置，可以导入生成的电影。显微镜安装步骤已在<a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/3585/Figure_3.1_merged.pdf" target="_blank">文件3.1</a>总结。 </li></ol> 4。迁移:计算速度和位移的图像处理和分析在这个例子中，是通过图像处理SlideBo确定5，6 5.0软件。还有许多其他方案，如ImageJ的7图像处理和分析。 <ol><li>导入SlideBook来自不同领域的图像生成的文件在不同的时间点。转到菜单栏下图片&gt;导入&gt; 幻灯片书阀芯 &gt;选择所需的文件（ 图4a）。 </li><li>在这里，我们的目标是跟踪单个细胞的运动。这是可以做到使用自动跟踪​​或个人跟踪。在这个例子中，我们用手动跟踪，因为这有利于排除工件容易，这是自动跟踪更加困难。 </li><li>打开的SlideBook文件，其中我们要进行跟踪。下菜单:选择面膜 &gt; 粒子跟踪协议 &gt; 手册粒子跟踪议定书"（图4b）。将出现一个窗口，开始和结束的时间点（ 图4c）。 </li><li> begin来跟踪单个细胞的运动，通过点击每个细胞（核）的中心。跟踪约10个单元，从每部电影，至少有三个不同的电影，从不同的地点应选择大约30个细胞，使将会从一个实验分析。每次实验应重复三次。允许不偏不倚的态度，选择不同的位置，如从前面的一些细胞的细胞中间，弯道等点击"OK"，一旦进行跟踪。 </li><li>随机迁移可以通过测量速度，速度或位移等的变化，要分析的数据，根据菜单&gt; 选择面膜 &gt; 粒子跟踪粒子跟踪协议分析。选择跟踪参数描述如下:（ 图5a）: <ol><li>跟踪 - 您希望跟踪从下拉列表中选择参数。在这个例子中，我们已经使用， 中心区 （OB中心的坐标Ject的）。 </li><li>点击"跟踪，并继续"。一旦跟踪完成后，会自动前进到下一步。 </li></ol></li><li>选择所需的统计为每个路径，如位移和平均速度（ 见图5b）: <ol><li> 平均时速 -总行驶距离除以前往距离所需的时间。 </li><li> 总排量 -对象行驶总距离。 </li><li>点击"计算，并继续"如果你想进步的下一步，或者干脆选择"计算"，如果你想查看您的统计资料，而不去下一步。将生成的文件可以保存为文本文件，以后可以导出到Excel。 </li></ol></li><li>为了追踪物体使用自动跟踪​​协议，第一个面膜被创建。 <ol><li>选择面膜&gt;图像分割&gt;段的窗口会出现选择，直到达到阈值，这样九月从别人arating一个对象&gt;单击"应用&gt;确定（ 见图5c）。 4.5.1步骤是相同的​​。 </li><li>删除对象小于 - 检查此框有选择地删除对象而不是一个给定的尺寸较小。在编辑栏输入的大小和选择任一微米为单位的像素。这是用来消除经常由碎片引起的工件。 </li><li>最小的运动 - 进入时间点的最低数量，必须跟踪，以确定路径。 </li><li>最大的移动 - 输入您希望从任何给定的对象从一个时间点到下一个最大的运动。 </li><li>的数据进行分析的步骤是相同的​​手动跟踪（见步骤4.5.2至4.6.2）。 </li></ol></li></ol> 5。代表结果随机迁移分析的一个例子，作为量化的速度和位移（ 图6）。在导出文件ţØExcel中，数据是用一个盒子和晶须阴谋策划。使用Mann-Whitney检验进行统计分析，比较，总的测试和控制之间的位移和平均速度。测试样品显示平均速度比对照增加。测试样品显示与对照相比，在总排量的增加。 电影:控制和测试控制MDA-MB-231和测试的MDA-MB-231细胞接种在胶原过夜。时间推移视频显微镜是由奥林巴斯显微镜，耦合电磁滨松相机。图像采取随机迁移期间共有18个小时的时间间隔为1小时。请参阅控制和测试样本的随机迁移的电影1，电影2。 显微镜下的设置: <img alt="图1" src="/files/ftp_upload/3585/3585fig1.jpg" /> 图1。设置图像的多点<html>捕获。 <img alt="图2" src="/files/ftp_upload/3585/3585fig2.jpg" /> 图2描绘捕获控制的步骤。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/3585/3585fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/3585/3585fig3.jpg" /> 图3。如何保存多张图片。 dflinebreak"&gt; 迁移的图像处理和分析。 <img alt="图4a" src="/files/ftp_upload/3585/3585fig4a.jpg" /> 图4a。导入图像数据分析的步骤。 <img alt="图4b" src="/files/ftp_upload/3585/3585fig4b.jpg" /> 图4b。对数据进行分析，使用粒子追踪协议的步骤。 <img alT ="图4c"SRC ="/ files/ftp_upload/3585/3585fig4c.jpg"/&gt; 图4c。窗口描绘跟踪粒子的路径长度。 数据分析的步骤。 <img alt="图5a" src="/files/ftp_upload/3585/3585fig5a.jpg" /> 图5a。描绘的粒子跟踪协议参数的步骤。 <img alt="图5b" src="/files/ftp_upload/3585/3585fig5b.jpg" /> 图5b。选择路径统计进行分析。 <img alt="图5c" src="/files/ftp_upload/3585/3585fig5c.jpg" /> 图5c。步骤创建自动跟踪协议的面具。 <img alt="图6" src="/files/ftp_upload/3585/3585fig6.jpg" /> 图6。平均时速（ 图6a）和位移（ 图6b）绘制在晶须情节。测试样品显示了增长的速度和位移作为比较控制。 电影 电影1。控制细胞的迁移。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/3585/3585movie1.avi" target="_blank">点击这里查看电影</a> 。 电影2。测试细胞迁移。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/3585/3585movie2.avi" target="_blank">点击这里查看电影</a> 。 人烟稀少控制的MDA-MB-231和测试的MDA-MB-231细胞接种过夜的胶原蛋白。时间推移视频显微镜是由奥林巴斯显微镜，耦合电磁滨松相机。在18个小时的时间间隔在随机迁移的1小时图像。为控制和测试，请参阅随机迁移的电影。

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我们什么都没有透露。

实时随机的移民法能够准确，灵敏的细胞迁移的参数，如速度和位移分析。此方法不仅限于结束点的测量值，因此它是更多的量化。由于细胞在正常培养液血清培养基监测，它减少了有害影响较长的潜伏期在无血清媒体。它已被证明的格式和基质8，因此，这种方法可以用来研究不同基质对移民的影响，打破了细胞接触依赖的细胞迁移。 的关键步骤之一，包括适当的?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养液</td><td> Thermo Scientific的</td><td> SH30243.01 </td><td></td></tr><tr><td>胎牛血清</td><td>双子座生物产品</td><td> 100-106 </td><td></td></tr><tr><td> OPTI-MEM </td><td> Invitrogen公司</td><td> 31985-070 </td><td></td></tr><tr><td>胶原</td><td>屋宇署</td><td> 354231 </td><td></td></tr><tr><td>显微镜与活细胞室及自动XY平台控制器</td><td>奥林巴斯</td><td>奥林巴斯1X81 </td><td></td></tr><tr><td>环境控制腔</td><td> NEUE </td><td> NEUE活细胞商会</td><td>我们已通过适当的光学腔的顶部建立一个自定义的矩形玻璃板的顶部。长方形容纳许多在我们的实验中使用的多孔板TS。 </td></tr><tr><td>自动XY平台</td><td>预先</td><td>在此之前Proscan </td><td>这允许乘以选定的XY位置，在时间的推移显微镜精确的回报。 </td></tr><tr><td>相机</td><td></td><td>滨松电磁相机C9100 </td><td></td></tr><tr><td>软件</td><td>通常购买厂商，如奥林巴斯显微镜</td><td>幻灯片书5 </td><td></td></tr></tbody></table>

quantitative analysis of random migration of cells using time-lapse video microscopy

细胞迁移是一个动态的过程，这是非常重要的胚胎发育，组织修复，免疫系统的功能，与肿瘤的侵袭1，2。在定向迁移，细胞外趋化信号的响应，或响应内在线索3的基本动力机械提供快速移动。随机迁移发生时，细胞具有低固有的方向性，使细胞探索当地环境。 细胞迁移是一个复杂的过程，在最初的反应细胞，经过极化和中迁移的方向延伸的突起。传统的方法来衡量，如Boyden小室迁移实验迁移是一个简单的方法来衡量在体外 ，从而使一个终点的结果作为衡量迁移的趋化。不过，这种做法既不允许个体迁移参数的测量，也不允许到visualization的细胞在迁移过程中经历的形态学变化。 在这里，我们提出了一种方法，使我们能够监测细胞迁移，在实际使用视频时间 - 时间的推移显微镜。由于细胞迁移和侵袭癌的标志，这种方法适用于研究癌症细胞迁移和体外侵袭。随机迁移的血小板已考虑为血小板功能的4参数之一，因此这种方法也可以是血小板功能的研究很有帮助。此法的优点是快速，可靠，重现性好，不需要手机号码的优化。为了维持细胞生理适当的条件下，在显微镜配备CO 2供应和温控器。细胞的运动进行监测使用安装在显微镜定期相机拍照。细胞迁移可以通过测量平均时速和计算verage位移，这是由Slidebook软件计算。

Watch this Scientific Journal Video about Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy at JoVE.com

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

这种方法允许在不同的细胞迁移参数，如速度，位移和速度的实时性和定量测量细胞的监测。实时方式与传统方法不同的是，这不是基于端点的定量迁移测量;相反，它允许不同的参数，不断监测和计算。

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 ...

quantitative-analysis-random-migration-cells-using-time-lapse-video

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

16.7K 次观看.  LSU School of Medicine. 这种方法允许在不同的细胞迁移参数，如速度，位移和速度的实时性和定量测量细胞的监测。实时方式与传统方法不同的是，这不是基于端点的定量迁移测量;相反，它允许不同的参数，不断监测和计算。

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

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

拆装时间推移显微镜监测肌动蛋白

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells 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 ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

使用时间的推移显微镜可视化缺氧诱导假死<em&gt; C.线虫</em&gt;胚胎

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

颅面部发育中的SOX10可视化:使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

时间推移视频显微镜对EYFP-Parkin的聚集性评估作为蜂窝Mitophagy一个标记

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

无透镜视频显微术在黏附细胞培养动态定量分析中的研究

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

延时结构照明显微镜线粒体核素的特定标签

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...