这项工作得到了从法新社德拉国民RECHERCHE（ChoAD"）的资助。我们也感谢基金会的财政支持ICM（研究所杜Cerveau ET DE LA Moelle）。

1。 FCS的安装校准<ol><li>启动共聚焦显微镜，激光，计算机，孵化温度和CO 2控制。 </li><li>确保SPAD值（单光子雪崩二极管）和荧光过滤器内的SPAD非常适合你的样品。检查的SPAD时间同步。小心才开始您的FCS的软件，一旦你的SPAD设置为收购准备。 </li><li>准备一个新鲜的霍乱毒素-Alexa488解决方案，在PBS稀释至浓度达到1微克/毫升（17.5海里）。 </li><li>优化解决方案的使用显微镜的内部检测的共焦成像。 </li><li>切换点扫描模式，选择一个解决方案中的样本点。确保激光照射的持续时间足够长的时间来执行您的FCS的收购（&gt; 5分钟）。 </li><li>切换到外部检测的SPAD和FCS的软件。监视你的荧光信号，并确保它是很好穗特德的SPAD（足够的信号，但没有饱和，10 000 50 000计数/ s是罚款）。如有必要，修改z位置，增益和/或激光功率，以达到正确的荧光信号。 </li><li>购买10套30秒的测量。采集时间应为您的样品进行了优化（最好的采样和漂白问题之间的妥协）。 </li><li>分析数据（见第4步），以确保您有相应的自相关曲线在溶液中扩散的荧光物种（ 图2中方程）拟合后得到的扩散时间是正确的（约0.2毫秒）（ 图3）。 </li></ol> 2。脂筏标记的活细胞染色<ol><li>板聚-L-赖氨酸（1mg/ml的）成像前一天，以确保他们的附着力是正确的涂8 Labteks HEK293细胞。使用无酚红的培养基，以确保没有荧光信号的扰动。 </li><li>洗涤细胞的HBSS（汉克的缓冲盐溶液）的两倍。 </li><li>加入500μL的HBSS霍乱毒素Alexa488（1微克/毫升）/ BSA（0.1％）在37到30分钟的细胞°C。霍乱毒素绑定到神经节苷脂，要优先脂筏分区。 </li><li>洗涤细胞的HBSS（汉克的缓冲盐溶液）的两倍。 </li></ol> 3。 FCS数据采集活细胞<ol><li>放在显微镜舞台上染细胞。确保温度和CO 2条件是最优的，否则这可能会导致文物扩散倍。 </li><li>优化使用内部显微镜检测（ 图4）利息细胞共聚焦成像。 </li><li>切换点扫描模式，并选择一个点在的利息细胞的细胞膜。执行1-2细胞，以确保细胞是不移动在收购过程中的实况图像。 </li><li>切换到外部检测的SPAD和FCS的软洁具。监视您的荧光信号，并确保它非常适合的SPAD（足够的信号，但没有饱和，10 000 50 000计数/ s的罚款）。如有必要，修改z位置，增益和/或激光功率，以达到正确的荧光信号。如果这是不够的，你可能会考虑改变染色条件。 </li><li>获得10样品30秒的测量。由于大多数细胞是自体荧光的，你可以看到在第一次收购，在荧光减少，由于自体荧光的衰落。 </li></ol> 4。 FCS数据分析<ol><li>设置相关的时间间隔，并与FCS软件生成的自相关曲线。相关间隔通常会解析滞后时间最短的时间最长（测量统计变得越来越不足时，最大的相关时间接近总采集时间，最大延迟时间被限制到80％不等收购时间上PicoquaNT软件）。 </li><li>对于每个样本，导出相应的荧光波动和时间的函数自相关的文件。 </li><li>使用数据分析和拟合软件，如原产地临导入文件。 </li><li>每个样品，确保收购期间没有漂白，即荧光保持稳定在30秒。丢弃任何措施，显示漂白，因为这可能会造成人为的扩散时间较长。 </li><li>检查其余的FCS曲线的形状是正确的（ 见图5）。确定平均FCS曲线。 </li><li>适合用适当的数学模型（ 图2）曲线。这适合会给你的扩散时间（s）和分子扩散（ESE）扩散时间（s）的比例。 </li></ol> 5。代表结果与FCS的校准霍乱毒素Alexa488解决方案的一个例子如图3所示</STRONG&gt;。经过检查，荧光的各项措施作为一个时间的函数没有显示任何漂白（ 图3A），个人平均FCS的曲线进行了计算。意味着FCS的曲线拟合方程相应的各种扩散模型（ 图2中的例子）。经典考虑来确定一个合适的质量参数的测定系数R 2。越接近R 2为1，更好的契合。在这种情况下，最准确的模型，以适应平均FCS曲线是一个人口扩散的荧光分子在三个层面（ 方程1）在图2和图3B自由。适合的扩散时间为0.32毫秒。曲线拟合（ 图3C）和R 2系数（0.99906）的残值给予适当的质量估计。 例如霍乱毒素Alexa488染色HEK2 FCS的分析93细胞在图5所示。具有不同扩散时间的的多相平均FCS的曲线的形状揭示存在的荧光分子的数量。这条曲线的最合适的对应模型与荧光探针三个群体:两个阻碍扩散（扩散在膜平面的两维）和一个在三维空间中的自由扩散（ 图2和图5中的方程2） 。人口后者对应于膜面，即绑定或解除绑定膜目标外移动的荧光分子，达到通过分泌或回收通路的膜，或离开内吞膜。两膜扩散倍，相应的霍乱毒素绑定到神经节苷脂，2毫秒的分子（25％），对应于脂筏之外的扩散，75毫秒分子（50％），相应的脂筏的扩散。请注意，任何光漂白ING在采集过程中，会导致人工扩散时间较长，因此可能造成的偏见，对脂筏域GM1本地化。 <img src="/files/ftp_upload/3513/3513fig1.jpg" alt="图1" /> FCS的设置（修改从Marquer等的图片12）的示意图如图1。 <img src="/files/ftp_upload/3513/3513fig2.jpg" alt="图2" /> 图2。扩散模型和相应的使用，以适应自相关曲线的方程的例子。结构参数S可以写为= Z 0与z / W 0 0有效焦距半径沿光轴的1 / e 2强度和w0有效LA或双侧的焦半径的1 / e 2强度。从传统的点扩散函数（PSF）的测量，可以提取这些值。 <img src="/files/ftp_upload/3513/3513fig3.jpg" alt="图3" /> 图3。扩散的霍乱毒素溶液中Alexa488 FCS的校准时间的评估。一）荧光波动功能的时间为30秒的采集有代表性的例子。乙）平均获得装有式（1）30秒采集10个样品（见图2）C）曲线拟合残差自相关曲​​线。 <img src="/files/ftp_upload/3513/3513fig4.jpg" alt="图4" /> 图4。的HEK-293细胞染色霍乱毒素-Alexa488。细胞成像SP5的共聚焦显微镜内部488nm激光线（徕卡公司，韦茨拉尔，德国）。荧光收集与X60计划复消色差透镜油浸物镜之间500和650 nm。 <img src="/files/ftp_upload/3513/3513fig5.jpg" alt="图5" /> 图5。在HEK293细胞的细胞膜的扩散时间霍乱毒素Alexa488的评估。 a）平均自相关曲线拟合方程2（ 见图2）。乙）从曲线拟合残差。 （修改从Marquer等 12。）

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没有利益冲突的声明。

这里介绍FCS的方法，使脂筏的荧光探针在活细胞中的利益分割的敏感和快速分析。 FCS结合共聚焦显微镜，单光子计数的灵敏度的定位精度。 FCS和标准的生化技术之间的主要区别是，FCS使脂筏的目标，而不是相对分区的分区DRMS隔离或合作修补的情况下是绝对测定。 FCS的数据采集每个样品约5分钟（30秒10的收购），因此是相当迅速，比一般的生化技术。这个速度让时间有可能在脂质筏分区的按照扰动，可能导致药品加成。然而，自相关曲线的分析可以是一个有点棘手拟合不同型号都必须通过确定浏览最准确的。此外，在收购过程中的漂白有要避免的，因为这可能会导致文物。 在这里，我们描述了一个例子，我们可以划定脂筏的一种脂质分区:神经节苷脂（ 图5）。不会有这样的研究可能只能应用于蛋白质的生化程序与标准。 FCS是不局限于虽然血脂和蛋白质也可以使用，无论是用荧光配体（如转Alexa555结合转铁蛋白受体，已知位于外排上9），或作为荧光蛋白的融合表达标签（如APP的YFP的或9 BACE1-GFP，在阿尔茨海默氏病的两个关键蛋白球员）。因此，它可以被用于广泛的目标。此外，FCS可以实现不仅在细胞株，这里描述的，而且在9个小学文化。 关于霍乱毒素，你应该牢记它有五聚体总神经节苷脂的倾向，从而造成膜微域可以是从本地的脂筏。这就是你为什么不能用霍乱毒素colocalisation来确定，如果你感兴趣的蛋白质是内部或外部的筏。然而，膜蛋白，如APP-YFP的BACE1-GFP，弥漫脂筏在9毫秒，非常类似霍乱毒素（75毫秒）的观察与扩散的60-80倍。因此，霍乱毒素可以用来校准的设置和检查，你可以区分快速和慢速扩散倍。 FCS是也适用于许多其他应用13,14，如oligomerisation状态的一种蛋白质15或16配体/受体药理研究测定。它也可以加上其他显微镜技术，如全内反射荧光（TIRF）17,18或受激发射损耗（受激发射损耗）19。事实上，受激发射损耗FCS允许更准确测定脂筏分区19，但到现在为止，受激发射损耗显微镜仍然是昂贵的，因此并不普遍。 FCS的主要限制是低浓度的荧光（在纳摩尔范围内）和快速扩散时间的需要，所以，荧光不会离开前激发量的光漂白。评估扩散的蛋白质的互补技术，包括FRAP还原（荧光漂白后恢复）和ICS（图像相关光谱）技术（20审查）。 FRAP还原力，高强度的激光束光漂白的一个单元格，然后监测漂白是荧光恢复后的地区。这次经济复苏来自从非漂白地区荧光漂白面积扩散。因此可以提取从恢复动力学的扩散时间。与邻高浓度可用于FRAP还原f荧光，并且可以访问大范围扩散倍，从而使补充到FCS的。 FRAP还原已经被用来评估是否有荧光是majoritarily内部或外部筏21,22，但它仍然是一个相当大容量的方法，量化荧光扩散的内部或外部筏在感兴趣的领域中所占的比例。 ICS是一个FCS的，像素空间相关性的计算方法是在一个给定的图像23像素的成像模拟。它的优点是适合慢慢扩散的荧光探针的研究，但仅限于二维分析。这个限制被克服被称为时空ICS（STICS）24 ICS的发展。 STICS使堆栈的图像时空相关荧光探针，因而是一个强大的技术，虽然它需要大量的计算实施，并具有较低的时间分辨率比FCS。事实上，无论是ICS和STICS是有限的，以处理慢得多次一帧图像的采集时间。 ICS的另一个扩展称为ICS的栅格（英国皇家特许测量师学会），25,26可以快速扩散过程的分析，通过利用光栅扫描模式，在大多数商业共聚焦显微镜。英国皇家特许测量师学会是多才多艺的，因为它可以使用一个大型的扩散范围（微秒到毫秒），但其实施（扫描参数，数据处理），可以很累赘。我们没有发现在文献中的许多论文，报告使用ICS和其派生技术研究脂筏27-29。

<html><table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>霍乱毒素B亚基的网站488 </td><td> Invitrogen公司</td><td>的C-34775 </td><td>兆瓦（五聚）= 57千克/摩尔</td></tr><tr><td>共聚焦显微镜</td><td>徕卡</td><td> SP5的</td><td></td></tr><tr><td>孵化器温度和CO 2控制</td><td>生活影像服务</td><td>立方盒</td><td></td></tr><tr><td> SPAD值（单光子雪崩二极管） </td><td> MPD公司（微型光子器件） </td><td> PDM系列（100微米的敏感区） </td><td></td></tr><tr><td>高通488纳米过滤器</td><td> semrock </td><td> 488纳米，阻断边缘BrightLine长通滤波器 部分＃ails.aspx？= FF01-488/LP-25"目标="_blank"&gt; FF01-488/LP-25 </td><td></td></tr><tr><td> FCS检测单位</td><td> picoquant </td><td> picoharp 300模块</td><td></td></tr><tr><td>采集和自动相关软件</td><td> picoquant </td><td> SymPhoTime </td><td></td></tr><tr><td>拟合软件</td><td> OriginLab </td><td> OriginPro8 </td><td></td></tr></tbody></table>

determination of lipid raft partitioning of fluorescently-tagged probes in living cells by fluorescence correlation spectroscopy (fcs)

在过去15年中，细胞膜是不是均匀的，并依赖于微，以发挥其职能的概念已成为被广泛接受。脂筏中富含胆固醇和鞘脂膜微。他们发挥的作用，在细胞的生理过程，例如信号和贩运1,2，但也被认为是几种疾病，包括病毒或细菌感染和神经退行性疾病中的关键球员。 然而，它们的存在仍是一个争论的问题4,5。事实上，脂筏的大小已估计为20纳米左右6，远低于传统的显微镜（约200纳米）的分辨率极限，从而解除了他们的直接成像。到现在为止，用于评估蛋白质脂筏内的利益分割的主要技术洗涤剂耐膜（DRMS）抗体的分离与合作修补。尽管广泛使用贝科使用他们比较容易实现的，这些技术容易文物，从而批评7,8。因此，有必要克服这些文物，能够探测活细胞中的脂筏分区技术改进。 在这里，我们提出了一个敏感的荧光标记的蛋白质在活细胞的细胞膜脂质的脂筏分区分析方法。这种方法，被称为荧光相关光谱（FCS），依赖于在位于内部或外部的脂筏的荧光探针的扩散时间的差距。事实上，在人工膜和细胞培养证明，探测器将扩散速度比内致密的脂质筏9,10外。分钟的荧光波动来确定扩散时间，测量的时间在一个焦点量（约1 femtoliter）的功能，用共聚焦显微镜在位于细胞质膜（ 图1）。自相关曲线，然后从这些波动，并安装适当的数学扩散模型11。 FCS可以用来确定分割的各种探针，只要他们有荧光标记的脂质筏。荧光融合蛋白的表达或荧光配体的结合，可以实现荧光标记。此外，FCS可以使用不仅在人工膜和细胞系，而且在小学文化，描述最近12。它也可以用来追踪脂筏的动态分区后，除了药物或膜脂组成的变化12。

Watch this Scientific Journal Video about Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS at JoVE.com

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

描述一种技术来探测荧光蛋白在活细胞的细胞膜脂筏分区。它发生在位于内部或外部的脂筏蛋白的扩散时间的悬殊优势。收购可以进行动态控制条件或吸毒后。

脂筏的测定分区在活细胞中的荧光标记探针的荧光相关光谱（FCS）

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

determination-lipid-raft-partitioning-fluorescently-tagged-probes

Nanyang Technological University

Research

JoVE Journal

Biology

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

16.1K 次观看.  Hôpital de la Pitié-Salpêtrière. 描述一种技术来探测荧光蛋白在活细胞的细胞膜脂筏分区。它发生在位于内部或外部的脂筏蛋白的扩散时间的悬殊优势。收购可以进行动态控制条件或吸毒后。

视频: Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

测量单个活细胞中的mRNA转录的动力学

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

科研

生物学

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

绿色荧光蛋白标记的质膜蛋白质的扩散系数的简易测量采用K空间图像相关光谱

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

采用Caco-2细胞研究脂质运输由肠

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

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

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

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

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

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 ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

外周相关蛋白血浆膜分型的测定

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

通过总内部反射荧光显微镜与数量和亮度分析相结合，活细胞中细胞表面受体的显微化动力学

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...