This work was supported in part by NIH grants (1R01CA137494, R01CA132115, R01CA086072 to R.G.P.), the Kimmel Cancer Center NIH Cancer Center Core grant P30CA056036 (R.G.P.), a grant from the Breast Cancer Research Foundation, generous grants from the Dr. Ralph and Marian C. Falk Medical Research Trust (R.G.P.) and a grant from the Pennsylvania Department of Health (R.G.P.). In part this work was supported by an American Italian Cancer Foundation postdoctoral fellowship (G.D.) and Bioimaging Shared Resource of the Sidney Kimmel Cancer Center (NCI 5 P30 CA-56036).The Department specifically disclaims responsibility for an analysis, interpretations or conclusions. There are no conflicts of interest associated with this manuscript.

1.成纤维细胞的电穿孔用两种表达载体EYFP - 帕金和pDsRed2-美图<ol><li>生长在10厘米组织培养板使用的DMEM（Dulbecco氏改进的Eagle培养基）补充有10％胎牛血清，2毫摩尔/升L-谷氨酰胺，100U / ml青霉素介质永生化小鼠胚胎成纤维细胞，和100毫克/毫升链霉素在含有5％的CO 2，在37℃潮湿气氛。 <ol><li>在80％的细胞汇合，通过无菌吸弃完整的DMEM培养基并加入10毫升的无菌1×磷酸盐缓冲液（PBS）（80克NaCl，2.0g的氯化钾，14.4克Na 2 HPO 4 2.4克KH的2 PO 4和1升蒸馏水2 O，PH值:7.4）。 </li><li>无菌吸弃PBS，并加入1毫升胰蛋白酶EDTA 0.25％。在37℃下孵育该板直至细胞被分离（2 - 3分钟）。加入4 ml完全DMEM培养基，重悬细胞，并取出10 _6;细胞悬浮液的血球计数的湖 注:血细胞计数被设计为使得在一组的16角正方形的细胞数量相当于细胞×10 4个 / ml的数。 </li></ol></li><li>种子1×10 6细胞到10厘米组织培养皿前24小时到电穿孔过程。 </li><li>无菌吸弃完整的DMEM中，加入10毫升无菌PBS的。无菌吸弃PBS，并加入1毫升胰蛋白酶EDTA 0.25％。在37℃下孵育该板直至细胞被分离（2 - 3分钟）。加入4 ml完全DMEM培养基和悬浮细胞在一个15毫升管。 </li><li>使用冷冻离心机（4℃）纺细胞向下，在250×g离心5分钟。丢弃无菌吸上清，悬浮颗粒在1毫升无菌PBS。旋细胞向下，在250×g离心5分钟，4℃。 </li><li>丢弃无菌吸上清，加100微升Ø˚F解决方案组合为电（82微升电解决方案V +18μl的补充解决方案1）的细胞沉淀和移液器轻轻悬浮颗粒（欲了解更多详情，请参阅用户指南）。在细胞悬液中加入2微克EYFP - 帕金（激发/发射527分之514）和1微克pDsRed2-美图（激发/发射六百二十〇分之五百六十五）的。 </li><li>转移溶液至用一次性巴斯德灭菌反应杯（这两个工具都随试剂盒提供），使用预先设定的程序的NIH / 3T3的U-030（单脉冲，电压200伏，电容960μF，脉冲时间20毫秒电穿孔，脉冲数:1）。 </li><li>电穿孔后，立即在37℃下在含有5％的CO 2的潮湿气氛中加入500μl新鲜预热完全DMEM培养基和种子上将6cm组织培养板活成像同类细胞孵育它们24小时。 </li></ol> 2.时间推移​​视频显微镜<ol><l我&gt;设置在显微镜的腔室在37℃至使用前的温度。 </li><li>在这一点上，准备一个特定的实时成像介质进行与实验协议。准备实时成像介质如下;混合DMEM苯酚 - 自由补充有10％胎牛血清，2毫摩尔/升L-谷氨酰胺，100U / ml青霉素，和100毫克/毫升链霉素。在37℃的水浴中预暖实时成像介质。 </li><li>丢弃电穿孔的细胞无菌抽吸培养基并加入预热实时成像介质1毫升，使用P1000吸管，并在37℃下孵育该板30分钟。 </li><li>在板潜伏期，流入5％的CO 2在显微镜的腔室已经在37℃的稳定温度。 </li><li>放置板的电穿孔细胞进入显微镜的房间，避免重大变动或振荡。 </li><li>使用软件接口，以便同时检测荧光设置显微镜从由共转染载体编码的融合蛋白刘哲民信号，EYFP-帕金（励磁范围495〜510 nm，发射范围520至550nm，绿色）和pDsRed2-三刀（励磁最大558纳米和最大发射583纳米，红）。 <ol><li>打开时间推移视频显微镜软件。在上面的菜单中选择EYFP - 帕金和罗丹明对pDsRed2-三刀FITC。在上面的菜单中选择放大倍率（20X）。 </li></ol></li><li>使用软件界面，寻找既表达共转染载体的单细胞和注册的位置。直到最少10个细胞的收集对于每个实验条件（实验组）重复此步骤。 <ol><li>选择菜单"应用程序"，然后单击多维采集。在多维采集窗口选择所需的参数，如收购的数量，每个采集和记录单元的位置之间的时间间隔。点击"采集"开始采集过程。 </li></ol></li><li>启动基底采集既EYFP-帕金和采集图像每5分钟的15分钟的总时间间隔的DsRed2-三刀荧光信号。 </li><li>制备预热实时成像介质用浓度FCCP的比最终工作浓度高两倍（0.1 - 10μM，细胞类型依赖性的）。 </li><li>中断采集过程，并添加轻轻地加入1ml用FCCP预热实时成像介质的成用吸管P1000显微镜的室内板。 </li><li>如前所述，为3小时的总时间，使用所记录的位置重新开始获取过程。保存所有采集到的图像，以分析它们，当收购是在创建过程mitophagic的视频。 </li></ol> 3.分析<ol><li>收集的个人计算机上的所有在".TIFF"格式所获取的图像</li><li>与图形软打开图片洁具及定义时间间隔由线粒体引起的去极化与FCCP以显示进入线粒体膜帕金招募的第一个形象出现（图像采集每5分钟）。重复此分析在实验组中的每个小区。 </li><li>标签上与实验组名的电子表格的列的第一个单元。对于每个实验组，测量为帕金招募最少10个细胞的的时间间隔。作出帕金招募所计算的时间间隔的平均值，并定义为每个实验组的标准偏差（平均值±SD，N ​​= 10）。 <ol><li>整理成单个计算时间间隔列。每列包含N =实验组10次测量。 </li><li>选择从公式生成器菜单功能"平均"。选择列中的数据。选择从公式生成器菜单功能"标准差"。塞莱克拉列中的数据。 </li></ol></li><li>使用统计方法，比较以确定在实验组之间mitophagy的动态一个显著差施加适当的统计实验组。 （例如，两组=学生t-检验 ，三个或更多个基团= ANOVA加一个事后检验 ） </li></ol>

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The authors have nothing to disclose.

时间推移显微镜可以被定义为，能在一次观测中时间延长活细胞成像到细胞动力学的过长时间的观察的技术。这种方法是从简单的共焦或活细胞显微镜分辨，因为它允许观察者能够实时识别单个荧光标记的蛋白质，并按照其一个单一的活细胞内的动态。事实上，共焦显微镜可以使用荧光抗体容易识别免疫标记的蛋白质，但它不允许观察细胞，并在其实际环境中的分子事件。 时间推移的视频显微术研究主要用于在不同类型的细胞，如HEK293 16，初级神经元17和Hela细胞18监测分子过程，但有亦临床应用，如怀孕和预测非整倍体19,20。时间推移显微镜提供embryolog用约胚胎发育的临床妊娠的其他信息派，这是可能的，这信息可被用于改善我们选择可行的胚胎转移的能力。利用定时显微镜的其他优点包括胚胎的较少处理和其对中间的观察，这将减少在实验室19内的损失或污染的风险菜肴。 细胞能够通过mitophagy降解损坏和不正常​​的线粒体保持健康的线粒体动态平衡。缺陷mitophagy链接到帕金森氏病21，凋亡22，老化23和癌症24。 Mitophagy由泛素连接酶帕金和PINK1 21驱动。由于这一过程中保持一个健康的线粒体地位的重要性，我们发现它的重要研究动态使用时间推移视频显微镜本次活动。建议亲母育酚将提供线粒体稳态是如何不同的细胞的条件，包括在基因表达和蛋白质的功能变化下的影响的证据。 在这项研究中，我们通过瞬时转染电成纤维细胞。电穿孔方法在优化的电压持续几微秒到细胞悬浮液，以通过干扰膜的磷脂双层，以增加细胞膜的渗透性施加的电脉冲。这个动作将导致暂时的孔的形成。的膜通透性允许被引入到细胞25的DNA。我们选择了这个过程，瞬时转成纤维细胞，才能有两个质粒EYFP - 帕金和pDsRed2-美图更高的转染效率。实际上，电穿孔过程是约10倍，比化学转染26更有效。该图像采集过程可由两个EXTE受到影响RNAL因素;温度波动，振动，湿度和内部因素;的pH值，细胞运动性。电穿孔和荧光跟踪被认为是对本实验方案的成功的最关键的两个步骤。即使转染效率比其它方法高，电穿孔杀死〜的电穿孔的细胞的20％。另一个主要的问题是在监视步骤的图像的采集质量。由于蜂窝环境是动态的，在聚焦平面需要，以维持在整个采集过程中的焦点连续调整。 线粒体损伤可以通过膜的线粒体去极化和氧化应激所引起的。这些因素诱发mitophagic响应。证据表明，线粒体去极化能诱发选择性mitophagy来自光损伤的实验从而导致活性氧的产生和线粒体损伤27-30。为了促使线粒体纪念品brane去极化和mitophagic响应，FCCP已被用作protonophore 10。 FCCP是一种离子载体，使质子穿过脂双层诱导细胞膜去极化。我们使用这种方法诱导mitophagy跟着分子标记的动态。 FCCP的浓度是至关重要的，因为它可以是细胞毒性和促进细胞死亡。由于这种方法依赖于活细胞的观察，FCCP的最终浓度必须在一个细胞类型依赖性以诱导mitophagy而不触发，导致细胞死亡的机制来调节。 总体而言，我们认为mitophagy的动态，用EYFP - 帕金作为荧光标记下，将提供新的见解线粒体功能。经由mitophagy选择性除去线粒体的是在维持线粒体的质量，细胞活力和稳态的关键过程。这种实时成像的方法，他将LP了解健康，并强调条件下mitophagic过程。 现代方法进一步超出使细胞动力学的电影时间推移显微镜观察。由于越来越多的技术，流式细胞仪正在与成像技术集成了监视和测量细胞和亚细胞结构31动态活动这个边界是模糊的。

Macro autophagy is an intracellular process that involves the catabolic degradation of both damaged and dysfunctional cellular components, such as organelles and proteins for the purpose of either recycling or energy production. To initiate this metabolic process, the cell engulfs the damaged cellular components into a double-membrane structure, known as an autophagosome, which fuses with a lysosome and its content is degraded and recycled 1,2. There are two major types of autophagy, the non-selective and selective. The non-selective autophagy process occurs when the cell is under nutrient deprivation conditions and needs to scavenge for both essential nutrients and energy. However, selective autophagy occurs to mediate the removal of both dysfunctional/damaged organelles and proteins that otherwise could be toxic. One of the most studied selective autophagy process is the removal of mitochondria, termed mitophagy 1,3-5.
Mitochondria are the central organelles for cell metabolism and the primary source of adenosine triphosphate (ATP) via oxidative phosphorylation through the electron transport chain, fatty acid oxidation, and tricarboxylic acid (TCA) cycle. Moreover, mitochondria regulate reactive oxygen species (ROS) production and release proteins that participate in cell death pathways 6-8.
PTEN-induced putative kinase 1 (PINK1) and Parkin RBR E3 ubiquitin ligase (Parkin) are the key proteins implicated in the mitophagy process. Parkin can protect against cell death by keeping the cell healthy through mitochondrial quality control9. Upon the loss of mitochondrial membrane potential, cytosolic Parkin is recruited to the mitochondria by PINK1. This recruitment triggers the sequential events of mitophagy 10. There is a broad range of evidence that mitophagy is a fundamental mitochondria quality control process and abnormalities in this process drive disease 7. For instance, autosomal recessive Parkinson's disease has been associated with mutations in the genes that encode for Parkin and PINK1 (PARK2 and PINK1, respectively) 11. The quality control of mitochondrial health is essential for the removal of mitochondria that contribute to the accumulation of ROS12. Excessive presence of intracellular ROS can lead to damage of both nuclear and mitochondrial DNA (DNA and mt DNA, respectively).
Herein, we show a time-lapse video microscopy approach to follow the aggregation of Parkin after the induction of Parkin-mediated mitophagy in immortalized mouse embryonic fibroblasts via in vitro administration of carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP), an uncoupling agent. FCCP disrupts ATP synthesis by short circuiting protons across the outer mitochondria membrane and hence uncoupling oxidative phosphorylation from the electron transport chain 13. Triggering the depolarization of the mitochondrial membrane leads to the disruption of mitochondria and selective Parkin-dependent removal. Therefore, transfecting the cells of interest with an expression vector encoding Parkin fused with a fluorescent marker (enhanced yellow fluorescent protein, EYFP) can be used as a fluorescent tag to follow the recruitment of Parkin during the mitophagic process. In order to visualize the mitochondria, we co-transfected pDsRed2-Mito, which encodes red fluorescent protein (DsRed2) that contains a mitochondrial targeting sequence of cytochrome c oxidase subunit VIII (Mito). pDsRed2-Mito is designed for fluorescent labeling of mitochondria14. The time required for Parkin translocation into the mitochondrial membrane can be measured and gives an indirect measure of cellular health. For example, we can say that if a cell line knocked-out for a particular gene of interest shows either a faster or slower recruitment of Parkin after the induction of mitophagy by FCCP, that gene product would be a key player in order to keep the metabolic rates of the cell at the physiological status and prevent the development of diseases. Therefore, the time-lapse video microscopy provides a very powerful tool for both basic and clinical research applications in following the dynamic of labeled proteins during their molecular processes and understanding how these processes are affected during a pathological condition.

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		<tr height="35">
			<td height="35" style="height:35px;width:735px;">DMEM</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">10-013-CV</td>
			<td style="width:369px;">Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">PHENOL-FREE DMEM</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">17-205-CV</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">FETAL BOVINE SERUM</td>
			<td style="width:357px;">Sigma-Aldrich</td>
			<td style="width:276px;">F2442</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">L-GLUTAMINE</td>
			<td style="width:357px;">Gibco</td>
			<td style="width:276px;">25030</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">PENICILLIN/ STREPTOMYCIN</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">30-002-CI</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">EYFP-PARKIN EXPRESSION VECTOR</td>
			<td style="width:357px;">Addgene</td>
			<td style="width:276px;">23955</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">pDsRed2-Mito EXPRESSION VECTOR</td>
			<td style="width:357px;">Clontech</td>
			<td style="width:276px;">632421</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">NUCLEOFECTOR 2B DEVICE</td>
			<td style="width:357px;">LONZA</td>
			<td style="width:276px;">AAD-1001S</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">NUCLEOFECTOR FOR KIT R NIH/3T3</td>
			<td style="width:357px;">LONZA</td>
			<td style="width:276px;">VCA-1001</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">ZEISS AXIOVERT 200M INVERTED MICROSCOPE</td>
			<td style="width:357px;">CARL ZEISS</td>
			<td style="width:276px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Carbonyl Cyanide 4-(trifluoromethoxy)-Phenylhydrazone (FCCP)</td>
			<td style="width:357px;">Sigma-Aldrich</td>
			<td style="width:276px;">C2920</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">MetaMorph</td>
			<td style="width:357px;">Molecular Devices</td>
			<td style="width:276px;">Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:735px;">ImageJ</td>
			<td style="width:357px;">National Institute of Health</td>
			<td style="width:276px;">Experimental Builder</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

这里，我们显示的时间推移的视频显微镜如何是一个强大的技术，可用于遵循荧光标记的蛋白质的分子事件在单个小区。代表结果还表明，该技术是如何允许采集高质量的图像。当分子过程的图象，而得到的，我们有机会来分析他们以不同的方式。这里，我们分析的感应mitophagy过程，这是至关重要的分子过程，维持细胞稳态选择性地除去受损的线粒体的起始之间的时间间隔。 这种技术开创了广泛的应用范围。它可以用于监控宏蜂窝过程如细胞迁移或通过涉及具有待研究的过程中的关键分子的标记微分子事件中使用。 ○:保together.within页="1"&gt; 帕金介导Mitophagy 选择性帕金介导的清除受损和不正常的线粒体过程的总结在图1中，其中，通过解偶联剂FCCP诱发膜的去极化触发选择性帕金介导mitophagy，招募PINK1至线粒体外膜。之后，帕金被招募到受损的线粒体和PINK1进行交互。这种复杂的帕金，PINK1导致受损的细胞器的泛素化并将其纳入了自噬体，它与溶酶体融合。称为mitophagy该过程被认为是为了保持细胞环境健康和允许官能线粒体8,15的再生的关键代谢过程。 实验协议 核苷酸"FO:保持-together.within页="1"&gt; 图2示出了实验的协议和用于前和FCCP诱导mitophagy过程之后下列EYFP-帕金招募的显微镜设置的示意性表示。 帕金招聘消偏到线粒体膜 在图3A中，我们显示帕金分配前和后FCCP管理。在基底的时间点（BT5，BT10和BT15分钟）EYFP-帕金（绿色）在整个细胞均匀地扩散和线粒体网络（红色）似乎是良好互联。以下FCCP施用（FT = 0分钟）（ 图3B），我们观察到线粒体分馏（FT = 5分钟）。然而，EYFP - 帕金仍在扩散均匀虽然出细胞质中。 Mitophagy在55分钟后FCCP管理的刺激下，EYFP - 帕金是在受损的线粒体膜招募触发mitophagy工艺（FT = 55）。 <img alt="图1" src="/files/ftp_upload/53657/53657fig1.jpg" /> 图1.帕金介导Mitophagy的示意图。递减膜电位（膜电势）的发生损坏或不正常的线粒体，可以通过解偶联剂FCCP诱导。膜电位的损失会触发选择性帕金介导mitophagy，招募PINK1（红）到线粒体外膜。帕金（蓝色）是通过用PINK1直接交互招募到损伤线粒体。帕金的存在导致泛素化损坏的细胞器（黄色），并将其纳入了自噬体，它与溶酶体融合。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53657/53657fig1large.jpg" target="_blank">请点击此处查看该图的放大版本</a>。 <img alt="图2" src="/files/ftp_upload/53657/53657fig2.jpg" /> 实验方案的图2.显微镜设置和表示。（A）在协议部分中描述的实验事件概述。在报道的实验事件允许操作者瞬时通过电穿孔以遵循使用（B）中的FCCP诱导mitophagy过程建立时间推移的视频显微镜的方法共转染与EYFP-帕金和pDsRed2-三刀细胞。 BT =基础时间点; FT = FCCP时间点。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53657/53657fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53657/53657fig3.jpg" /> 图3.成纤维细胞显示帕金Recruitme<html> NT后线粒体膜去极化由FCCP。EYFP -帕金（绿色）易位进入线粒体（红色）诱导被时间推移视频显微镜基础的时间点（BT）在监测和发布FCCP管理（FT）。从BT = 0，EYFP - 帕金和线粒体荧光检测每5分钟。 （A）EYFP -帕金被均匀在整个基础期扩散和线粒体网络是否完好（白色箭头）。由FCCP（B）线粒体分离，由于膜的去极化记录在FT = 5分钟（白色箭头）和EYFP -帕金募集到受损的线粒体膜开始在FT = 55分钟（白色箭头）。白色框，表示面板（5X）的放大区域。有关本实验方案的应用程序的更多详细信息，请参阅参考8。 （比例尺:10微米）。&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy at JoVE.com

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

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

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.

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 ...

time-lapse-video-microscopy-for-assessment-eyfp-parkin-aggregation-as

Nanyang Technological University

Research

JoVE Journal

Biology

7.1K 次观看.  Thomas Jefferson University, SKCC. 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.

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

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 ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

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

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

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 ...

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 ...

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, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

一种用于阴南芥叶片安装的通用方法

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...