This project was supported by grants NIH NIGMS 8P41 GM103540-28 and P50-GM076516

1，样品制备<ol><li>保持在使用补充有10％胎牛血清和100 IU / ml青霉素链霉素50微克/毫升的组织培养瓶中的CHOK1细胞。培养细胞在5％二氧化碳培养箱在37℃。 </li><li>嘉实然后板CHOK1细胞在14毫米直径的微孔与0.16毫米的表面厚度。种子细胞用于成像的最佳密度，周围60-70％汇合。 </li><li>过夜孵育细胞，在37℃，5％CO 2。在HBSS（汉克缓冲盐溶液）中含有50nM的Lysotracker DND26绿色和150纳米的微管蛋白的跟踪绿色的溶液中洗涤细胞三次，并孵育细胞。孵育细胞1小时，在37℃。可选步骤:孵化前，用线粒体基质染色的染料（25纳米）执行额外的污点。 </li><li>在HBSS洗涤细胞三次，以除去任何未结合的染料。前IMAG加入新鲜的DMEM培养基荷兰国际集团的细胞。 </li></ol> 2，显微镜配置显微镜在视频协议中描述的粒子跟踪组装市售的倒置显微镜（ 图4）的框架上。三维轨道粒子跟踪然而商业模块现已上市。 <ol><li>使用相干变色龙超二钛萨飞秒激光激发光源，与690之间的NM-1040 nm的可调输出波长范围。 </li><li>确保激光束在显微镜的后部端口使用的IR-涂覆的镜子对准。激光束被使用旋转半波片之后是方解石线性偏振衰减。衰减的光束，使得在样本的平均功率为0.5〜2毫瓦。 注:该激光束由一对检流计电机致动反射镜，使在样品平面中聚焦光束的位置和轨迹的控制反映。在典型配置的准直激光束出射的检流计反射镜被扩展（10X）穿过光束扩展器，在短通分色镜反射后进入显微镜物镜的后前。 </li><li>通过将高数值孔径物镜水（60X，NA 1.2）插入光路收集来自样品的荧光的光。根据在一个或两个信道配置所需的发光波长选择在荧光滤光块。采用发射带通滤波器，以进一步选择所发射的荧光的光谱范围。 </li><li>将样品放置到电动阶段，并调整显微镜物镜的使用目的压电控制器的微调。 </li><li>直接从过滤立方体进入光电倍增管信号在那里被歧视，送到数字I / O数据采集卡的光。 注:电脑产生的波形是I / O卡的模拟输出，并provid编辑的扫描仪控制电子设备。从光电倍增管和输出信号提供给扫描仪的光子计数输入被测量，并且经由I / O卡的实验室荧光动力学SimFCS软件控制。 </li></ol> 3，影像学<ol><li>定位在舞台上的细胞，并集中使用透射光照明。 </li><li>切换到光栅扫描成像，以确定已将染料的细胞，以可视化的底层微管。确定初始区域囊泡运动存在。 </li><li>一旦分离的囊泡被识别时，设定为轨道跟踪参数如下: <ol><li>选择的轨道半径，根据该颗粒的大小被跟踪来定义圆形扫描的大小。对于一个点发射器，设置在轨道等于激发束以最大化灵敏度和响应的点扩散函数（PSF）的腰部的半径。 </li><li>设置轴向距离，以定义在执行本地化沿轴向的粒子位置两个轨道之间的距离。设定为1.5-3倍的PSF腰部的轴向距离。 </li><li>根据颗粒来设定使用了上各点的轨迹，这也决定了光漂白速率的时间的亮度定义的停留时间。使用10-100微秒之间的停留时间。 </li><li>点中每个轨道的数目设置为64或128，以产生轨道周期为4-32毫秒的顺序和用于确定粒子的位置提供了一个高的时间分辨率。 </li></ol></li><li>改变直流发送到反射镜，以通过光栅扫描图像中的光标选择通过图形用户界面，以围绕所述颗粒的光束偏移信号。 </li><li>首先从开关光栅成像显微镜模式，以轨道扫描跟踪。 </li><li>激活的反馈和数据收集。 </li></ol> 4 TrajectORY分析<ol><li>使用该软件，以显示在沿着轨道和在一个"强度地毯"的形式中，每个时间点的每个点所收集的荧光。沿着所述地毯收集的强度提供了与其它明亮物体的粒子之间的相互作用的信息。使用软件（在X，Y扫描仪且z -piezo的时间，即直流偏移）以及来自光电倍增管中的时间序列的形式收集的随着时间的荧光强度，以显示两个轨迹信息。 </li><li>使用时间序列的代表性和"强度地毯"的信息来选择的轨迹的兴趣只是一个区域。 </li><li>使用该软件，显示粒子轨迹的所选部分的二维投影。根据颗粒的荧光强度选择适当的控制，以颜色编码的轨迹。 </li><li>选择要显示在p选项根据荧光强度的文章轨迹在3D和彩色编码的。旋转用控制形象化沿微管的溶酶体运动的特征的轨迹在三维。 </li></ol>

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	<li>So, P. T., Dong, C. Y., Masters, B. R., Berland, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+photon+excitation+fluorescence+microscopy.">Two photon excitation fluorescence microscopy.</a> Annu Rev Biomed Eng. 2, 399-429 (2000).</li><li>Dupont, A., Lamb, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+three+dimensional+single+particle+tracking.">Nanoscale three dimensional single particle tracking.</a> Nanoscale. 3, 4532-4541 (2011).</li><li>Estrada, L. 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The authors have nothing to disclose.

尽管荧光显微技术及仪器，在过去几年取得的荧光粒子在三维空间运动轨迹具有高时间分辨率的巨大进步一直保持在该领域的挑战。如果高时间分辨率已经在两个维度上被实现跟踪颗粒，延伸到轴向方向通常带来大幅减少了系统10的频率响应。 在这个视频协议，我们重点跟踪单个粒子在三维空间中活的细胞中利用双光子显微镜的高时间分辨率（32毫秒在这个实验中）的示范应用。需要进行这样的实验设置通常是可用的，以执行激光扫描显微镜实验室。都是乐器插件的3D轨道跟踪和控制软件可在市场上，对于commercia升激光扫描显微镜，如那些由Olympus生产的。使用的脉冲的高功率红外激光源，其中可用的，是在实验装置，因为它不需要去扫描发射的荧光的设计有利的。此外，它被证明在过去的红外线照射是朝向细胞更良性的，在特定条件11下，给定的聚焦荧光和光致漂白的减少。 虽然可以使用灵敏照相机进行两维的荧光粒子的快速跟踪，由于没有任何三维信息的可以是常限制在解释的轨迹信息的生物物理含义。例如，水疱沿微管运动经常碰到的微管交叉区域8,12。在这种情况下，使用了三维跟踪技术克服了采用三维结构的2D投影的限制。 由于我们的目标纳米定位的压电器件的时间响应，在z轴移动的期间被限制在大约30毫秒，而在x，y方向上它可以深入到几毫秒。该方法的定位精度秤作为信噪比4,5。该方法的唯一明显的局限性， 即颗粒的环境的全局视图的损失，由重建的时间强度的地毯13，跟踪荧光颗粒的相互作用的历史与邻近的荧光来源的可能性补偿显示可以是相同或不同的光谱发射。 我们表明，溶酶体的运动，通过两种驱动蛋白和动力蛋白经受定向沿微管运动，动力小泡，可以跟活的细胞中使用市售的染料染色后。近日，溶酶体的二维跟踪是科尔兴高采烈的微管网络的超分辨率成像，在检测到的囊泡采取在微管的微管的交点8的路由的努力。溶酶体的3D轨迹显示比单向运动更为复杂的行为。虽然平均定向运动可以观察到，弯曲，扭转和沿细管可以旋转可以从这些轨迹进行观察。 我们已经确定在执行这些实验中的两个关键步骤:首先，它是必要的，以实现即在同一时间足够高，以获得明亮的染色颗粒的溶酶体标记密度，而同时提供了颗粒低的密度足以使单个颗粒的跟踪。在两个相交的颗粒的情况下，在显微镜可从一个粒子切换到另一个的同时跟踪。第二个关键步骤涉及的像素停留时间的选择，可以让一个合理的平衡是吐温高信噪比和跟踪速度。我们已观察到，在每个跟踪周期32毫秒的顺序跟踪速度通常足以跟随溶酶体发生在低于1微米/秒的速度快速定向运动。 总之，轨道跟踪允许在三个层面，并与&lt;40毫秒荧光标记的囊泡的胞内运动的时间分辨率来衡量。三维轨道跟踪具有在将来被用于不同的高度动态的细胞过程，如我们的染色质动力学的实时转录和等离子体激元纳米颗粒在细胞内介质的扩散研究的研究的可能性。

大量方法已被开发出迄今为止为在使用显微镜的三维跟踪荧光发色粒子。大多数方法依赖于使用的快速照相机，非常适合以跟踪在两个维度上，典型地结合了镜的出射光学系统的定制修改，以实现跟踪在轴向方向上。激光扫描显微镜（或者共聚焦或两个光子）通常可以追踪的荧光粒子通过进行的Z-堆栈的时刻的顺序，尽管这种过程通常是耗时的，并且会产生合理的时间分辨率（10赫兹）只有当被跟踪的粒子是由有源反馈机制2保持在一个小的光栅成像区域的中心。 锁入到该颗粒的想法是在轨道的跟踪方法的基础。取而代之的是光栅扫描的圆形轨道围绕的荧光粒子进行。荧光沿强度轨道精确本地化粒子的位置4。 该颗粒的局部位置然后可用于致动显微镜扫描器和重新中心在粒子位置的轨道。显微镜的检流计扫描仪是由一个模拟电压驱动。这个电压的AC分量允许用聚焦激光束进行的轨道， 即正弦波和施加到X和Y扫描反射镜将允许执行一个圆形轨道上的余弦波。直流信号的偏移有助于改变轨道中心的位置。一旦一个轨道期间被确定并且对于交流信号的波形构成，所述反馈系统需要更新信号的唯一的直流分量。 软件能够读取从检测器收集到的荧光信号是必需的，以控制扫描仪和检测器，来计算粒子的位置和更新的直流断设定。对于一个成功的反馈成像仔细选择的轨道参数（大小和定时）的需要和这些参数具有基于所述荧光体颗粒，有必要进行跟踪的特性来进行调整。 该技术的物理和数学基础，在过去4,5 2D和3D应用程序进行了描述。在这个协议中，我们简要地描述了安装的主要硬件部件，并且更详细的参数的选择和样品制备所需的典型的实验中，使我们以下溶酶体中的活细胞内的高时间分辨率的位移。

<table><tbody><tr><td>Lysotracker DND 26</td><td>Life Technologies</td><td>L-7526</td><td></td></tr><tr><td>Tubuline tracker Green</td><td>Life Technologies</td><td>T34075</td><td></td></tr><tr><td>Mitotracker RED</td><td>Life Technologies</td><td>M7512</td><td></td></tr><tr><td>Coherent Chamelon- ultra II TI</td><td>Coherent</td><td></td><td></td></tr><tr><td>Glan Taylor Calcite Polarizer</td><td>Melles Griot</td><td>03PTA001</td><td></td></tr><tr><td>Galvanometer-motor mirror</td><td>Cambridge Technologies</td><td>M 6350</td><td></td></tr><tr><td>Dichroic mirror</td><td>Chroma Technologies</td><td>700 DCSPXR</td><td></td></tr><tr><td>Motorized stage</td><td>ASI</td><td>MS2000</td><td></td></tr><tr><td>Piezo&amp;nbsp;</td><td>PI</td><td>P721-LLQ</td><td></td></tr><tr><td>Photomultiplier tube</td><td>Hamamatsu</td><td>H7422P-40</td><td></td></tr><tr><td>Data acquisition card</td><td>IO tech</td><td>PCI 1128-4000</td><td></td></tr><tr><td>Imaging software</td><td>LFD</td><td>Global for images-SimFCS</td><td></td></tr></tbody></table>

3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles

这个视频协议的目的是讨论如何使用修改后的双光子显微镜1执行和分析三维荧光轨道粒子跟踪实验。相对于传统的方法（光栅扫描或基于一个堆栈帧的宽域），三维轨道跟踪允许定位和跟踪具有高空间（10 nm的精度）和时间分辨率（50赫兹的频率响应）的三维位移移动荧光粒子的长度尺度数百微米2的。该方法是基于一种反馈算法来控制，以围绕该对象执行的圆形轨道上的双光子激光扫描显微镜的硬件要跟踪:反馈机制将保持在中心的荧光物，通过控制的位移扫描光束3-5。为了证明这项技术的优势，我们跟着快速移动的细胞器，溶酶体，内人艾尔文细胞6,7。细胞根据标准方法镀，并用市售溶酶体染料染色。我们简要讨论的硬件结构和详细的控制软件，以执行在活细胞内的3d轨道的跟踪实验。我们详细讨论，以控制所述扫描镜和使该光束的动作在颗粒周围的封闭轨道所需要的参数。我们得出结论:通过展示如何此方法可有效地用于活细胞内跟踪沿在三维微管标记的溶酶体的快速运动。溶酶体可以用移动的速度在0.4-0.5微米/秒的范围内，通常显示沿微管网8定向运动。

根据这一协议，快速的3D单粒子跟踪可以在里面用改进的双光子显微镜来跟踪荧光标记的溶酶体的位移活细胞中进行。进行实验包括跟踪一个孤立的溶酶体内体成熟过程后，9移动单元格中的。溶酶体用荧光绿色染料染色，激发在930 nm的利用双光子激发。我们的数据表明，有可能获得的x，y和z的位移轨迹具有32毫秒（ 图1a）的时间分辨率。地毯分析显示沿各轨道上的时间（ 图1b）记录的强度。期间的跟踪发射的荧光的积分强度可以被记录（ 图2a）。重建的三维轨迹显示的定向运动，如从泡囊移动的微管网络上可以预料的。 ádditionally，能够关联的三维轨迹的周边区域（ 图2b）的光栅扫描的图像。这证实了沿微管束的方向，颗粒的运动。 将细胞染色，另外一个红色的标记线粒体其移动过程中记录这些细胞器的囊泡的最终互动。有可能相关的细胞（ 图3a）内的粒子的三维轨迹从mitotracker荧光发射（ 图3b）记录的强度地毯。这使我们能够记录囊泡的接近相互作用的线粒体网络的小区内的位置的函数。记录在"线粒体通道"发光峰值不给出关于这样的细胞器相互作用的功能的信息。它可以提供关于他们的相互影响只是时间信息和被跟踪的相对于该粒子的荧光物体的相对空间位置。这个相对位置，从那里发光峰值被记录在轨道内的角度萃取。我们用来执行该实验的显微镜的示意图在图4中可以观察到。 <img alt="图1" src="/files/ftp_upload/51794/51794fig1highres.jpg" /> 图1位移与时间的轨迹和强度的地毯。一） 的x，y和z溶酶体位移与时间的关系的痕迹。 二）强度的轨迹（ 即沿着每个圆形轨道竖直轴线所收集的强度是时间）提供了关于颗粒的荧光环境信息。的盒装外亮点对应于与其他明亮的天体粒子的相互作用，并在轨迹的日相关联的跳跃<html> ê轨道上最亮的荧光源重新中心。盒装区域对应于轨迹的部分在两跳之间。 <img alt="图2" src="/files/ftp_upload/51794/51794fig2highres.jpg" /> 图2:继溶酶体的三维轨迹。收集利用粒子跟踪一）3D溶酶体的轨迹的一部分重建。该轨迹是颜色编码为从粒子收集到的荧光强度（红色，高，蓝色，低光子计数）。轴刻度是纳米。注意，X，Y，Z轴有不同的范围。的三维轨迹的二）2D投影，在成像中的栅格的微管立即扫描的顶部显示该轨迹的覆盖的轨迹被收集。 三）线粒体染色的mitotracker后。 再3"SRC ="/文件/ ftp_upload / 51794 / 51794fig3highres.jpg"/&gt; 图3扩展的轨道跟踪到一个以上的通道（激发波长930纳米）。一）细胞。 二）绿色通道（收集此通道的荧光强度地毯内的溶酶体的三维轨迹，用于跟踪）。 三）在红色通道，其中线粒体沾满了红色荧光强度矩阵地毯有针对性的染料。其中的轨迹变得与线粒体（箭头）相接触的区域出现在地毯强度凸点，当光束周围溶酶体轨道跨越进入线粒体基质。 <img alt="图4" src="/files/ftp_upload/51794/51794fig4highres.jpg" /> 图4示意图实验装置:改良的双光子显微镜 。的激光设置示意图:2光子钛萨激光射程690-1,040纳米是我们编，偏振器和一个半波片，用于控制的激励功率。所有其他项目列在使用指定缩写的数字。我们使用了60X的目标与1.2 NA。

Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用:细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

3d-orbital-tracking-modified-two-photon-microscope-an-application-to

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.2K 次观看.  University of California, Irvine. In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope. 

视频: 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

内吞体动力学在生活神经末梢与四维荧光成像可视化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

科研

神经科学

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

可视化 果蝇 胚胎中含脂质细胞器的细胞骨架依赖性运输

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

发育生物学

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

生物工程

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

在体内果蝇前运动神经末端的单分子跟踪

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

高速超分辨速度显微镜在活原纤毛中的应用

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

生物学

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

酵母的4D显微镜

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

三维细胞外囊泡的直接随机光学重建显微镜

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

Source: Tsuruta, F., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55488">Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes.</a> J. Vis. Exp. (2017).
This video demonstrates how neuronal vesicles are labeled with specific fluorescent proteins to visualize their movement. First, a plasmid coding for a fluorescent protein is incubated with a transfection agent and then introduced into a culture of neurons. Subsequently, images of the neurons are acquired and analyzed to track the vesicles&#39; movement.

使用荧光成像可视化神经元中的囊泡运动

1. Imaging Vesicle Motility

	Prepare the plasmids that will label each type of vesicle. For instance, use EGFP-Rab5 for early endosomes, EGFP-Rab7 for ...

3d轨道跟踪的改进双光子显微镜之应用:细胞内的囊泡的跟踪

摘要

探索更多视频

此视频中的章节

内吞体动力学在生活神经末梢与四维荧光成像可视化

可视化果蝇胚胎中含脂质细胞器的细胞骨架依赖性运输

一种实时3D 单粒子跟踪协议

使用荧光探针定量培养的神经元中的内体和溶酶体运动

在体内果蝇前运动神经末端的单分子跟踪

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

高速超分辨速度显微镜在活原纤毛中的应用

酵母的4D显微镜

三维细胞外囊泡的直接随机光学重建显微镜

使用荧光成像可视化神经元中的囊泡运动

3d轨道跟踪的改进双光子显微镜之应用:细胞内的囊泡的跟踪

摘要

探索更多视频

此视频中的章节

内吞体动力学在生活神经末梢与四维荧光成像可视化

可视化 果蝇 胚胎中含脂质细胞器的细胞骨架依赖性运输

一种实时3D 单粒子跟踪协议

使用荧光探针定量培养的神经元中的内体和溶酶体运动

在体内果蝇前运动神经末端的单分子跟踪

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

高速超分辨速度显微镜在活原纤毛中的应用

酵母的4D显微镜

三维细胞外囊泡的直接随机光学重建显微镜

使用荧光成像可视化神经元中的囊泡运动

可视化果蝇胚胎中含脂质细胞器的细胞骨架依赖性运输