我们的工作是支持的生物技术和生物科学研究理事会。我们想感谢我们恳请提供CFP-LC3的表达质粒吉森TAMOTSU教授。

1。细胞的制备<ol><li>种子的低传代次数的HEK-293T细胞稳定表达GFP-DFCP1对22毫米的圆形盖玻片的Dulbecco修饰老鹰的培养基（DMEM）中培养细胞过夜，至30-40％汇合（大致目标为80％汇合后2天 - 一天的活细胞成像）。 </li></ol> 2。细胞转染<ol><li>准备转染复杂的混合，每块板，含100微升的OptiMEM的我减少血清培养基，3微升X-tremeGENE的DNA转染试剂和0.5微克pECFP-LC3质粒DNA。上下吹打，轻轻混匀，在室温下孵育15分钟。 [注:我们已经多次发现其他脂质体2000转染试剂，比如，有很多毒性，此外，他们还与许多显微镜技术对自己的干扰产生的荧光颗粒。 </li><li>吸介质板和补充新鲜DMEM预温在37℃ </li><li>转录sfection复杂的细胞通过移液，孵育细胞24小时。 </li></ol> 3。细胞孵育的细胞器标记（可选） <ol><li>添加Mitotracker有/ lysotracker的DMEM培养液的最终浓度为75 nM，并保持在冰上，用铝箔覆盖在隼管，沿整个实验天，以避免曝光和重复的冷冻和解冻循环。 </li><li>取出2毫升的Mitotracker有/ lysotracker的含培养基的等分试样，并在37℃下预热，从转染细胞中吸出培养基，更换Mitotracker有/ lysotracker的含培养基孵育细胞30 - 60分钟。 </li></ol> 4。制备的活细胞成像的孵化室（图1） <ol><li>清洁金属和塑料O型圈，用75％乙醇和应用硅润滑脂，金属O型圈的边缘上。 </li><li>使用镊子除去盖玻片从板干燥介质从盖玻片的底侧的多余的避免介质与油脂混合太多，因为这会增加泄漏的可能性。 </li><li>离开盖玻片塑料O型圈的窗台上休息，适合金属O型圈顶部的塑料O型圈，与盖玻片夹在中间，以创建一个封闭的腔。 </li><li>从这点上，无缓冲剂，避免长时间培养的细胞在DMEM培养基中，以防止pH值的变化，以发生，DMEM中的碳酸氢盐缓冲系统需要人工的CO 2浓度顶起来的腔室与从板的介质;为5-10％，环境空气中的CO 2浓度要低得多。 </li></ol> 5。饥饿的细胞<ol><li>将孵化室显微镜舞台上。 </li><li>完全培养基吸出洗涤用2ml饥饿培养基中的3倍，以确保没有氨基酸的DMEM培养液，最终抑制自噬反应仍然设置计时器。 </li></ol> 6。显微镜<ol><li>将需要一个适当的成像系统，其被配置在活细胞的宽视场落射荧光。这通常包括研究级倒置显微镜框架，激烈的广谱光源，反射镜和过滤特定的荧光蛋白（S）/染料（S）的利益，一个高品质的物镜，一个敏感的CCD / SCMOS相机和孵化室。所有主要的显微镜制造商提供完整的宽视场系统适合活细胞成像，但它也有可能以家庭建立一个系统，使用各种厂家的组件和控制它使用开放源码软件如微经理（ <a href="http://valelab.ucsf.edu/~MM/MMwiki/" target="_blank">HTTP: / / valelab.ucsf.edu /月/ / MMwiki</a> ）。在我们看来，最重要的方面是使用高灵敏度的系统，这样的表达水平的荧光记者可以保持在最低限度。 </li><li> SEL阿拉斯适当的细胞图像。 <ol><li>选择大而扁平的细胞，将允许更多的自噬体形成事件被捕获。此外，已经开始生产更大数量的omegasomes的的选择的细胞。 </li><li> 30分钟后开始视频捕获或到自噬响应，以争取更大的自噬体形成比例，每部影片的事件。 </li></ol></li><li>成像。 <ol><li>使用高倍率镜头（100X 1.4 NA）。 </li><li>调整的激发光的强度最大的10-20％，以防止光漂白。 </li><li>设置相机（滨松ORCA ER，像素大小为6.45微米）到100-500毫秒曝光，2x2的分级和100的增益。 </li><li>设置每10秒1帧图像采集率。 </li></ol></li></ol> 7。自噬体形成ImageJ的活动创建蒙太奇 [这是可以做到在一个非系统的方式，通过简单地扫描为电子合并后的视频通气孔的兴趣，但它也可以是系统化的，如下面所述。] <ol><li>打开图像栈3（或2）捕获ImageJ的/斐济的渠道。 </li><li>应用绿色，红色和蓝色的LUT（查找表），相应的通道;合并3种颜色，并保存。 </li><li>从"分析"选项卡中，选择"工具"&gt;"ROI经理... &gt;设置，然后选择一个随机区域定义大小的堆栈中的第一张图像。 </li><li>从"图片"选项卡，选择"复制"，以便复制所选区域的堆栈。 </li><li>从"图片"选项卡，选择"堆栈"&gt;蒙太奇...创建一个试探性的蒙太奇包含的所有帧抓获。扫描所有帧中的蒙太奇，一个完整的自噬体形成事件的第一个和最后一个帧事件的记录。 </li><li>从"分析"选项卡中，选择"工具"&gt;"ROI经理...选择在第一幅图像的堆栈对其他2种颜色，以及合并后的颜色图像和同一地区重复堆叠。 </li><li>从T他文件"选项卡上，选择"新建"&gt;"图像"，并设置宽度根据初步选定像素使用ROI经理，设置高度的数量4倍的高度设置在ROI经理加上3个像素之间的颜色和空间合并，并设置在每个堆栈的帧的数量的片。 </li><li>从"图片"选项卡，选择"堆栈"&gt;"工具"&gt;"插入...，然后插入一个在另一个之上，每个子协议栈之间留下1个像素的空间。 </li><li>从"图片"选项卡，选择"堆栈"&gt;蒙太奇...创建一个蒙太奇的起点和终点与第一个和最后一个帧的自噬体形成捕获的事件。 </li></ol>

<ol>
	<li>Mizushima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+process+and+function.">Autophagy: process and function.</a> Genes Dev. 21, 2861-2873 (2007).</li><li>Mizushima, N., Yoshimori, T., Ohsumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Atg+proteins+in+autophagosome+formation.">The role of Atg proteins in autophagosome formation.</a> Annual review of cell and developmental biology. 27, 107-132 (2011).</li><li>Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+from+phenomenology+to+molecular+understanding+in+less+than+a+decade.">Autophagy: from phenomenology to molecular understanding in less than a decade.</a> Nat. Rev. Mol. Cell Biol. 8, 931-937 (2007).</li><li>Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+revisited:+a+conversation+with+Christian+de+Duve.">Autophagy revisited: a conversation with Christian de Duve.</a> Autophagy. 4, 740-743 (2008).</li><li>Yla-Anttilba, P., Vihinen, H., Jokitalo, E., Eskelinen, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tomography+reveals+connections+between+the+phagophore+and+endoplasmic+reticulum.">3D tomography reveals connections between the phagophore and endoplasmic reticulum.</a> Autophagy. 5, 1180-1185 (2009).</li><li>Hayashi-Nishino, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subdomain+of+the+endoplasmic+reticulum+forms+a+cradle+for+autophagosome+formation.">A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation.</a> Nat. Cell Biol. 11, 1433-1437 (2009).</li><li>Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+in+vivo+analyses+of+cell+function+using+fluorescence+imaging+(*).">Emerging in vivo analyses of cell function using fluorescence imaging (*).</a> Annu. Rev. Biochem. 80, 327-332 (2011).</li><li>Mizushima, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+autophagosome+formation+using+Apg5-deficient+mouse+embryonic+stem+cells.">Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.</a> The Journal of Cell Biology. 152, 657-668 (2001).</li><li>Itakura, E., Mizushima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+autophagosome+formation+site+by+a+hierarchical+analysis+of+mammalian+Atg+proteins.">Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins.</a> Autophagy. 6, 764-776 (2010).</li><li>Axe, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagosome+formation+from+membrane+compartments+enriched+in+phosphatidylinositol+3-phosphate+and+dynamically+connected+to+the+endoplasmic+reticulum.">Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum.</a> J Cell Biol. 182, 685-701 (2008).</li><li>Walker, S., Chandra, P., Manifava, M., Axe, E., Ktistakis, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+autophagosomes:+localized+synthesis+of+phosphatidylinositol+3-phosphate+holds+the+clue.">Making autophagosomes: localized synthesis of phosphatidylinositol 3-phosphate holds the clue.</a> Autophagy. 4, 1093-1096 (2008).</li></ol>

没有利益冲突的声明。

在这个协议中所描述的方法允许自噬体的形成过程中的蛋白质的定位的可视化。我们已经尝试了各种方法，可视化描述的事件，包括点扫描共聚焦，旋转盘共聚焦和全内反射荧光显微镜（TIRF）。我们发现，一般用途的标准宽视场落射荧光灵敏度和分辨率之间的最佳折衷。这可确保良好的信号噪声，最小photo-bleaching/photo-toxicity和快速收购。光学切片的缺乏不是一个问题，如果被选择的单元格的适当的区域的图像， 即细胞传播和平面的外周。然而，重要的是，所使用的成像系统被适当地配置（无论是在所用的硬件和系统设置）。 为了获得最佳的空间分辨率，建议使用高倍率，H室内运动场数值孔径的油镜（水浸泡镜片将提供接近盖玻片没有任何好处成像）。这是建议平衡照明的强度（ 例如，与中性密度过滤器），相机的设置（曝光时间，分箱和增益），以最大限度地提高的信号与噪声和减少漂白。这将不得不凭经验，但作为指导，使用100倍的1.4 NA透镜时，我们通常会降低我们的激发光的功率最大的10-20％，并设置摄像头（滨松ORCA ER，像素大小为6.45微米）至100-500毫秒曝光，2x2的分级和100的增益。 图像采集速率应设置在1帧每1-10秒的范围内。获取更高的帧速率的图像将确保更好的图像（更好的时间分辨率）之间的连续性，但会暴露细胞更多的光线，从而提高photo-bleaching/photo-toxicity""。 如果成像fluorescenc的ê渠道，它必须保证，捕获通道之间的延迟最小化（减少曝光时间，适合快速过滤兑换）。这将减少出现在合成图像中的运动伪影的机会。如果运动伪影被证明是难以避免的考虑使用图像分路器（装置，方便两个荧光通道使用一个摄像头同时采集）或双摄像头适配器。 ，成像蓝色通道需要选择合适的过滤器和镜子，以防止交叉排放的蓝色荧光的绿色通道。我们已经成功地使用了一个的奥林巴斯CellR显微镜，使用至多彩V照明灯，使特定波长，带宽和强度的选择，所以是非常灵活的。然而，光源是一些白色的光来选定wavelengths.For除了这个原因，我们安装在带通激发滤光片（多）&#39;漏&#39;多维数据集，所以有额外的激发光的过滤。下面结合镜/过滤器使用（所有Semrock）。对于GFP mCherry:励磁FF01-479-585，发射器FF02-525/40（GFP）和FF01-607/36（mCherry），分色镜FF505/606-Di01的。对于CFP励磁，发射FF01-523/610，分色镜FF440/520-Di01的FF01-416/501。我们也使用一个的不同CellR显微镜，这给次优的结果，交叉发射蓝色荧光的绿色通道。这种显微镜使用白色光源，并且具有快速的滤光轮来选择激励wavelengths.This装置的波长的选择是有限的在车轮8的过滤器，但有一个单独的滚轮来调节强度。下面结合镜/过滤器被使用。对于GFP（Semrock）:励磁FF01-470/40号号，发射FF02-525/50，分色镜FF495-DI02。对于:CFP和mCherry::励磁FF01-427/10（CFP，Semrock）572/23（mCherry色度），发射FF01-472/30中，（CFP，Semrock）632/60（mCherry色度），，，分色镜89006bs（色度）。 与其他成像技术相比，这种技术的意义是双重的:第一，它可以捕捉的本地化的蛋白在活细胞中的兴趣，和第二，它可以增加加入的第四维时间中提取的信息。然而，与外源蛋白质总是定位错误的可能性，要么是由于表达水平的增加，或由于标记，因此，活细胞成像应结合免疫染色，固定细胞中的内源性的POI，为了证实结果。最后，值得注意的是，为了增加分析的空间分辨率，活细胞成像，可结合免疫EM。

自噬是一个高度动态的过程，这就需要大量的蛋白质，协调的最终结果自噬体形成1-3。显微镜的技术可能是最常用的应用为研究自噬4。大多数自噬蛋白的本地化已被广泛地研究了在固定的细胞，通过免疫染色的内源性蛋白和荧光标记的外源蛋白质的表达。此外，电子显微镜（EM），单独和组合使用免疫金标，精致的细节描 ​​述了这些结构5,6。尽管这些技术已经建立了一个事实，即我们了解自噬体形成的3个维度空间，他们并没有提供足量的4 个维度的信息-时间。活细胞成像克服了这个障碍，因为它允许自噬体的形成可能接近ble到实时7。这种技术最早是研究自噬吉森和同事8，并已被越来越多地用于今后。 时间推移显微镜捕获的POI在活细胞中，并经过一段时间的本土化。通过比较这些信息与一个特征的自噬和/或细胞器标记活细胞成像分析，可以把兴趣点，在更大的空间和时间范围内自噬体的形成。活细胞成像分析是基于POI本地化沿自噬体的形成的所有步骤的重复捕获，而固定细胞成像是基于一个单一的捕获。因此，活细胞成像可以证明在自噬体形成的具体步骤的POI的贡献，而固定细胞成像，只能承担角色的POI，其平均定位的基础上同时捕获许多自噬在不同阶段其lifecyCLE。 虽然活细胞成像分析能力高的方法，它具有一些固有的局限性，这应该加以考虑。首先，活细胞成像的要求中的一个或多个外源性荧光标记蛋白的表达。荧光标记往往是体积大，它们有时可以改变一种蛋白质的行为，由于空间位阻的原因。这种情况加剧膜蛋白的，因为他们需要在有限的空间发挥作用的2维膜。值得注意的是，自噬体膜的结构，因此它们的形成需要大量的膜相关蛋白。 另一些问题是连接到该POI的表达水平。原则上，应表达的外源蛋白质与内源性蛋白的水平。这确保了其亚细胞定位的重要调节器将不饱和的，和日E解析生物相关。此外，应该避免的，自噬蛋白的过度表达时，他们都表达了以上的内源性水平，他们往往会抑制自噬反应9。相反，因为POI的表达水平应该是很高的，足以让一个很好的一段时间后，其本土化没有光漂白，要达到一个妥协。实现最佳的外源蛋白质在哺乳动物细胞中的表达水平，需要微调的特定的，但它是可行的，通过建立和筛选稳定表达细胞系的不同水平的POI。 即，可以实现与标准的荧光显微镜的空间分辨率是另一个限制因素。分辨率可能被限制为一定数量的原因，但在最好的情况下，将横向分辨率在250 nm附近。这意味着，比这更小的距离分隔的任何对象会出现连接（或作为一个单一的将代表大于他们实际上是在图像对象），小于250纳米的物体。因此，图像应始终被解释考虑到这一点和互补的技术，如EM，将需要解决精美的超结构的细节。 最后，活细胞成像的本质要求露出光电池，有可能在很长的一段时间。这可能会改变细胞的生理反应，这种现象被称为光毒性。 我们已成功地用于活细胞成像的PI3P结合蛋白DFCP1的第一次来形容，自噬来源于富含PI3P环状结构称为omegasomes，这是密切相关与ER股10,11。我们已经清楚地表明，LC3阳性结构开始形成在与omegasomes密切相关。在这里，我们建议，用人的细胞系稳定表达GFP-DFCP1活细胞成像的感兴趣的蛋白质，建立了一个强大的空间和时间的帧的表征，它的作用在自噬体的形成。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41965</td>
			<td></td>
		 </tr>
		 <tr>
			<td>OptiMEM I</td>
			<td>Invitrogen</td>
			<td>31985-062</td>
			<td></td>
		 </tr>
		 <tr>
			<td>MitoTracker Red FM</td>
			<td>Invitrogen</td>
			<td>M22425</td>
			<td></td>
		 </tr>
		 <tr>
			<td>LysoTracker Red DND-99</td>
			<td>Invitrogen</td>
			<td>L-7528</td>
			<td></td>
		 </tr>
		 <tr>
			<td>X-tremeGENE 9 DNA Transfection Reagent</td>
			<td>Roche Applied Science</td>
			<td>6365787001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>22 mm coverslips</td>
			<td>VWR</td>
			<td>631-0159</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 mm plates</td>
			<td>Fisher NUNC</td>
			<td>153066</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Silicon grease</td>
			<td>RS Components Ltd. </td>
			<td>RS 494-124</td>
			<td></td>
		 </tr>
		 <tr>
			<td>O-rings</td>
			<td></td>
			<td></td>
			<td>Custom made</td>
		 </tr>
		 <tr>
			<td>Attofluor Cell Chamber</td>
			<td>Invitrogen</td>
			<td>A-7816</td>
			<td>Suggested alternative to custom-made O-rings</td>
		 </tr>
		 <tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td>Inverted microscope</td>
		 </tr>
		 <tr>
			<td>Objective</td>
			<td>Olympus</td>
			<td>UPLSAPO 100XO</td>
			<td>N.A. 1.4, W.D. 0.13, FN 26.5</td>
		 </tr>
		 <tr>
			<td>Camera</td>
			<td>Hamamatsu</td>
			<td>ORCA-R2 C10600 10B</td>
			<td>Progressive scan interline CCD</td>
		 </tr>
		 <tr>
			<td>Illuminator</td>
			<td>TILL Photonics</td>
			<td>Polychrome V</td>
			<td>Ultrafast monochromator</td>
		 </tr>
		 <tr>
			<td>Incubation chamber</td>
			<td>Solent Scientific</td>
			<td>Cell^R IX81</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software</td>
			<td>Olympus</td>
			<td>SIS xcellence</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

live cell imaging of early autophagy events: omegasomes and beyond

自噬细胞反应缺乏的营养物质，特别是氨基酸的情况下触发。自噬是指由形成的双层膜结构，称为自噬体，即隔离细胞质中，寿命长的蛋白质和蛋白质聚集体，有缺陷的细胞器，甚至是病毒或细菌。自噬体最终导致大量降解它们的内容与溶酶体融合，所产生的营养成分被再循环返回到细胞质。因此，细胞自噬是细胞的动态平衡至关重要，自噬失调可导致疾病，最显着的神经退行性疾病，衰老和癌症。 自噬体的形成是一个非常复杂的过程，其的细胞已分配一组特定的蛋白质，被称为核心自噬机械。是该核心自噬机械的功能辅以不同的细胞过程中所涉及的额外的蛋白质， 如在MEMBRANê贩运，线粒体和溶酶体生物学中。自噬体的形成和降解的协调，这些蛋白质构成的高度动态和复杂的响应的自噬。活细胞成像允许按照每个自噬相关蛋白的分子贡献水平的自噬体形成一个单一的事件，并实时的，因此该技术提供了一个高时空分辨率。 在这里，我们使用的细胞系稳定表达GFP-DFCP1的，我们的分析建立了空间和时间的情况下。 DFCP1的痕迹omegasomes，易制毒化学结构导致自噬体的形成。可以打上一个红色或青色荧光标记蛋白质的兴趣点（POI）。不同的细胞器，如急诊室，线粒体和溶酶体，都参与自噬体的形成在不同的步骤，并且可以使用一个特定的跟踪染料标记。时间推移显微镜AUTOPhagy在这个试验性设置，使得要被提取的信息有关的第四维， 即时间。因此，我们可以按照自噬在空间和时间上的POI的贡献。

描述的协议，我们已经按照本地化CFP标记LC3稳定的细胞系表达GFP标记DFCP1的自噬诱导条件下，使用时间推移显微镜。这个实验的结果是2系列或成堆的图像，从绿色和蓝色通道之一，对应GFP-DFCP1和CFP-LC3的捕获。我们已进一步分析这些视频使用ImageJ，在以对应于单一的自噬体形成的协议部分中所描述的事件，创建蒙太奇。这种分析使我们能够证明LC3阳性的自噬体来源于一个阳性DFCP1的omegasome。在蒙太奇图2中所示，形成一个omegasome从第二帧，在一个小点的形式显现。的omegasome开始扩大，以形成特征的环形结构，6分钟后达到其最大的直径。接下来，开始omegasomeÇollapsing并最终消失，约10分钟后。现在把LC3阳性结构的形成或自噬体在的omegasome形成的上下文中，我们观察到自噬体出现后omegasome，约1.5分钟后变得清晰可见。自噬体开始扩大的omegasome，第一点，然后环紧密结合，并开始崩溃时omegasome自噬体的芽。自噬体最终留下的，显然是为了与溶酶体融合，后omegasome消失。这一分析提供了一个明确的指示约2结构之间的函数关系，根据LC3阳性自噬源于相应omegasomes。 在另一个例子中，我们已经添加了一个的溶酶体跟踪为了捕捉到时间和空间的关联，形成自噬体与溶酶体（ 图3 </strong&gt;）。事实上，几分钟后崩溃omegasome，开始新生的自噬体与溶酶体相关联，并最终与溶酶体融合。自噬体与溶酶体的融合很大程度上CFP-LC3荧光淬灭，想必由于CFP标签在酸性环境中的溶酶体曝光。 然而，相同类型的分析可以产生难以解释的结果，由于各种各样的原因。在该示例中， 如图4所示，结果变成不可解释的，由于在焦点的漂移。视频开始成功地捕捉自噬体的形成，但是焦点漂移发生后3分钟大关。捕获的视频将继续在接下来的6分钟的重点，，手动更正后的9至10分钟大关，但最终的焦点。虽然这种分析，使得无法区分是否形成自噬体事件时捕获的重点是纠正的这几乎完成后，最初的事件或之后开始一个新的，在焦点的漂移。 的其他问题都可以归结到汇合的培养细胞。例如，当细胞生长在高于最佳汇合，它们被强制扩大相邻小区的顶部，这使得聚焦相当具有挑战性。此外，在高汇合生长的细胞，往往会被强调，增加饥饿的启动前的背景水平的自噬活性。 最后，荧光相关的问题相当普遍。 CFP相比，GFP荧光活性较弱，经常会在稍后阶段的视频捕获光漂白。这样的视频分析可能会导致假阴性的结论形成自噬体的空间关联的POI。但是，这些问题是可以克服的通过使用如mTurquise2变种之一。另一种常见的现象学Ñ​​源于红色标记的荧光的事实，在较低的pH值的溶酶体是不灭的。许多自体吞噬蛋白生理完成其生命周期进入溶酶体，而的自噬最终与溶酶体融合。此外，非功能性的蛋白质往往是对象在溶酶体降解。因此，人们可能会结束后，自噬体与溶酶体的非关联性，而不是一个实际的物理协会之间的红色标记的POI和omegasomes。 <img src="/files/ftp_upload/50484/50484fig1.jpg" alt="图1"/> 图1。孵化室。塑料O型圈紧密配合金属O型圈的边缘和底部薄窗台向内延伸。盖玻片放在窗台上的塑料O形环，，那么塑料O形环安装在底部的金属O型圈。通过这种方式，盖玻片砂之间wiched 2 O型圈，创造一个封闭的腔。 <img src="/files/ftp_upload/50484/50484fig2.jpg" alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50484/50484fig2highres.jpg" /> 图2。细胞表达GFP-DFCP1的和CFP-LC3被饿死30分钟，每10秒1帧的速率和成像阿蒙太奇自噬体的形成有代表性的事件。从绿色和蓝色通道的信号是伪彩色相应的绿色和红色。箭头表示第一个明显的omegasome和自噬。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50484/50484fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/50484/50484fig3.jpg" alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/50484/50484fig3highres.jpg"/> 网络连接GURE 3。培养细胞表达的GFP-DFCP1和CFP-LC3，红色与lysotracker，饿死30分钟，在1帧，每15秒的速度拍摄。自噬体的形成有代表性的事件是阿蒙太奇。绿色，红色和蓝色通道的信号是伪色绿色，蓝色和红色的相应。箭头表示第一可辨别的omegasome和自噬。箭头表示第一次捕捉自噬体与溶酶体融合。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50484/50484fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/50484/50484fig4.jpg" alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/50484/50484fig4highres.jpg"/> 图4。细胞表达GFP-DFCP1的和CFP-LC3被饿死30分钟，每10秒1帧的速率和成像。蒙太奇子邻的一个例子<html>捕捉提出ptimal。从绿色和蓝色通道的信号是伪彩色相应的绿色和红色。箭头表示第一个明显的omegasome和自噬。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50484/50484fig4large.jpg" target="_blank">点击这里查看大图</a> 。 视频1。图2给出了自噬体形成事件的视频回放速率为每秒4帧。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50484/50484movie1.avi" target="_blank">点击这里观看电影</a> 。 视频2。在图3中的自噬体形成事件的视频，箭头表示第一，形成的omegasome，和第二，自噬体与溶酶体融合。回放速率为每秒4帧。"&gt;点击这里观看电影。 视频3。图4中的自噬体形成事件的视频回放速率为每秒4帧。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50484/50484movie3.avi" target="_blank">点击这里观看电影</a> 。 表1中。列表中的特定的试剂和设备所需的协议，以及与相应的供应商和产品目录号。 缓冲器<table border="1"><tbody><tr><td fo:width=".5in"> 缓冲区 </td><td fo:width="1.5in"> 组成 </td><td fo:width=".5in"> 步骤二手 </td></tr><tr><td>饥饿中等</td><td> 20毫米HEPES pH值7.4 </td><td> 5.2 </td></tr><tr><td></td><td> 140 mM氯化钠</td><td></td></tr><tr><td></td><td> 1毫米氯化钙2 </td><tD&gt; </td></tr><tr><td></td><td> 1毫米氯化镁2 </td><td></td></tr><tr><td></td><td> 5毫米葡萄糖</td><td></td></tr><tr><td></td><td> 1％BSA </td><td></td></tr></tbody></table> 表2中。在这个协议中使用的缓冲区。列表中使用的缓冲液，它们的组合物和它们在协议中使用的第一个步骤，在该步骤中列出。

Watch this Scientific Journal Video about Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond at JoVE.com

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

时间推移显微镜荧光标记的细胞自噬标记允许自噬反应具有高时空分辨率的动态监测。使用特定的自噬和细胞器标记在3种不同颜色的组合，我们可以按照自噬体形成在一个强大的空间和时间上下文的蛋白质的贡献。

活细胞成像早期自噬活动:Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

live-cell-imaging-of-early-autophagy-events-omegasomes-and-beyond

Nanyang Technological University

Research

JoVE Journal

Biology

18.4K 次观看.  The Babraham Institute. 时间推移显微镜荧光标记的细胞自噬标记允许自噬反应具有高时空分辨率的动态监测。使用特定的自噬和细胞器标记在3种不同颜色的组合，我们可以按照自噬体形成在一个强大的空间和时间上下文的蛋白质的贡献。

视频: Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

科研

生物学

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

小鼠中自噬激活有氧运动

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

活细胞成像血小板脱水和分泌流量

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

基于细胞健康监测的实时细胞成像分析对低密度脂蛋白胆固醇的吸收检测

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...