Name: mapping molecular diffusion in the plasma membrane by multiple-target tracing (mtt)
Uploaded: 2012-05-27T14:00:00
Description: Watch this Scientific Journal Video about Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT at JoVE.com

我们感谢我们的团队成员提供技术援助，特别是管委会Blache，以及M Irla和乙Imhof，对他们的支持和富有成效的讨论。通缩和分娩的数字再现自然方法提供。支持这个项目是由国家科学研究中心（INSERM）和马赛大学，体制补助和特定地区研究所国家杜癌症，法新社国立德拉Recherche（普罗旺斯 - 阿尔卑斯 - 科特迪瓦-d&#39;Azur补助的ANR-08-PCVI 0034-02，国家情报局2010汪曾祺1214 01）基金会争取，LA RECHERCHEMédicale（队报labéliséeFRM-2009）。 VR是支持从法甲国立CONTRE LE癌症的奖学金。

在这段视频中，我们提出了一个完整的单粒子追踪实验，利用量子点定位到一个特定的膜受体。这项实验的主要目标包括在鉴别不同类型的测量活细胞的细胞膜内的分子扩散行为。事实上，在膜产生的分子运动，通常可以从布朗扩散偏离线性指示或局限在nanodomains 26日至29日，例如。我们的目标，同时为许多技术上是可行的受体后，提供了在一个活细胞的细胞膜内发生的动力学产生的各种?...

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旁边的细胞和显微镜的各个方面，在单粒子跟踪，分析代表了相当一部分的工作。这解决了算法用于执行三大任务：检测，评估和重新连接了每帧的山峰。但随之而来的这项工作方面驻留在拟订算法本身，这可能需要适应任何新的专门调查，基本上最后，额外的步骤（如破译运动模式，相互作用或化学计量学）。 然而，一旦该算法充分开发，运行，它是简单的，特别是因为?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td><td> 数量 </td></tr><tr><td> COS-7细胞系</td><td> ATCC </td><td> CRL-1651 </td><td> 5000细胞/孔</td></tr><tr><td>无钙的HBSS中2 + </td><td> GIBCO公司</td><td> 14175 </td><td> 1毫升</td></tr><tr><td>胰蛋白酶EDTA的0.05％ </td><td> GIBCO公司</td><td> 25300 </td><td> 1毫升</td></tr><tr><td> 8以及实验室TEK </td><td> NUNC </td><td> 155441 </td><td> 1 </td></tr><tr><td> QDot-605链霉</td><td> Invitrogen公司</td><td> Q10101MP </td><td> 20毫米</td></tr><tr><td colspan="4"> 生物素厂 （为晶圆厂合成，见参考文献21） </td></tr><tr><td>从单克隆抗体的Fab 108 </td><td> ATCC </td><td> HB-9764 </td><td> 200微克</td></tr><tr><td> NHS-生物素<html></td><td> Thermo Scientific的</td><td> 21435 </td><td> 18.5微克</td></tr><tr><td colspan="4"> 完全培养基 </td></tr><tr><td> DMEM培养液</td><td> GIBCO公司</td><td> 41965 </td><td> 500毫升</td></tr><tr><td>胎牛血清</td><td>西格玛</td><td> F7524 </td><td> 50毫升</td></tr><tr><td> L-谷氨酰胺</td><td> GIBCO公司</td><td> 25030 </td><td> 5毫升</td></tr><tr><td>肝素钠</td><td> GIBCO公司</td><td> 15630 </td><td> 5毫升</td></tr><tr><td>丙酮酸钠</td><td> GIBCO公司</td><td> 11360 </td><td> 5毫升</td></tr><tr><td colspan="4"> 成像介质 </td></tr><tr><td>与钙的HBSS中2 + </td><td> GIBCO公司</td><td> 14025 </td><td> 25毫升</td></tr><tr><td>肝素钠</td><td> GIBCO公司</td><td> 15630 </td><td> 250μL </td></tr></tbody></table>1“&gt; <tbody><tr><td> 设备 </td><td> 公司 </td><td> 参考 </td></tr><tr><td>倒置显微镜</td><td>尼康</td><td>日食TE2000U </td></tr><tr><td>日光灯</td><td>尼康</td><td> intensilightÇ-HGFIE </td></tr><tr><td> 1.3适用100X目标</td><td>尼康</td><td>计划氟1.30 </td></tr><tr><td> 1.49不适用100X目标</td><td>尼康</td><td>亚洲生产力组织的TIRF 1.49 </td></tr><tr><td>相机</td><td>罗珀科学</td><td>级联512 B的</td></tr><tr><td>恒温箱</td><td>生活影像服务</td><td>包装盒</td></tr></tbody></table>附录：例如MTT的补充分析脚本 ，功能 MTT_example（FILE_NAME） ％％％Basic示例显示如何恢复MTT法的输出结果 ％％％绘制每个TRACe和建立直方图 荧光强度的％％ nargin &lt;1％，如果没有提供FILE_NAME？ 文件= DIR（&#39;* STK&#39;）; 如果的isEmpty（文件），DISP（&#39;没有数据&#39;在当前目录）， 返回，结束 FILE_NAME =文件（1）名称;％默认：第一STK文件 DISP（[&#39;使用&#39;FILE_NAME&#39;默认&#39;]） 结束 file_param = [FILE_NAME“_tab_param.dat&#39;];％的输出文件 ％负载数据 CD％或（“output22）（”output23），根据版本使用 声明：版本2.2只产生7个参数， ％一个额外的参数，噪声，2.3版本中添加了 ％在一个表中一次读取所有的参数， ％tab_param = fread_all_param（file_param）; ％TAb_i = tab_param（2:8结束，:); tab_j = ... ％在不同的表读取所有参数（除frame_number） ％[tab_i，tab_j，tab_alpha，tab_radius，tab_offset，tab_blk，tab_noise] = fread_all_data_spt（file_param）; tab_i = fread_data_spt（file_param，3）;％，指数是3，因为跟踪号码和车架号码，非知识性，被丢弃！ tab_j = fread_data_spt（file_param，4）; tab_alpha = fread_data_spt（file_param，5）; tab_blk = fread_data_spt（file_param，8）; ％％循环过的痕迹 n_traces =大小（tab_i，1）; ％表是由N_frames colums N_traces线 ITRC = 1：N_traces no_blk_index = tab_blk（ITRC，:)&gt; 0％，非只有闪烁的步骤 情节（tab_i（ITRC No_blk_index）的，tab_j（ITRC No_blk_index）的） ＆NBSP; xlabel（&#39;I（像素）&#39;），ylabel（&#39;J（像素）“） 标题（[微量＃&#39;num2str（ITRC）]） DISP（&#39;请取得任何未来跟踪的关键“），暂停 结束 ％FLUO直方图的 n_datapoints = SUM（tab_blk（:)&gt; 0）;％，无闪烁的步骤 * SQRT（N_datapoints）的）历史（tab_alpha（tab_blk&gt; 0），2％使用2sqrt（N）的垃圾箱 xlabel（&#39;强度（非盟）&#39;），ylabel（&#39;发生&#39;） 标题（“粒子的荧光强度的直方图&#39;）

mapping molecular diffusion in the plasma membrane by multiple-target tracing (mtt)

我们的目标是获得细胞膜发生在不同的生物功能的分子过程的全面描述。我们的目标是在描述复杂的组织和细胞膜的动态，在单分子水平，开发的分析工具，专门在高密度单粒子追踪 （SPT）： 多目标跟踪 （MTT）1。电视显微镜，单分子提供毫秒级和纳米级分辨率1-11，允许膜组织12-14详细表示，通过精确映射，如细 ​​胞受体的定位，流动性，禁闭或相互作用的描述。 我们重新审视六方会谈，实验和算法。实验方面包括：优化设置和细胞标记，以达到尽可能高的标签密度特别强调，为了提供一个分子动力学动态快照就这样发生在膜。算法的问题有关的每一步都用于重建的轨迹：峰值检测，估计和重新从15,16图像分析处理，通过特定的工具。实施后，检测通缩允许抢救峰最初隐藏相邻，峰强。值得注意的是，提高检测直接影响重联，减少内轨迹的差距。已评估使用各种标签密度和噪声值，这通常代表两个主要局限在高时空分辨率的并行测量的蒙特卡罗模拟表演。 17纳米的精度得到单分子，无论是连续使用开/关photoswitching或非线性光学，可以提供详尽的意见。这是19,20风暴18日 ，棕榈，RESOLFT 21 STED 22,23，WHI等17 nanoscopy方法的基础上CH可能常常需要固定的样品成像。中心任务是有限的单分子所产生的衍射峰的检测和估计。因此，提供足够的假设，如恒定的定位精度，而不是处理布朗运动，MTT法是直截了当地适用于纳米级的分析。此外，MTT法可以从根本上被使用在任何规模：不仅为分子，同时也为细胞或动物，例如。因此，MTT法是一个功能强大的跟踪算法，发现在细胞和分子尺度的应用。

Watch this Scientific Journal Video about Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT at JoVE.com

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

多目标跟踪是一个自制的算法跟踪单独标记分子在活细胞的细胞膜。有效地检测，估计和跟踪在高密度的分子随着时间的推移，提供一个用户友好，全面的工具来研究纳米膜动力学。

测绘在细胞膜分子扩散，多目标跟踪（MTT）

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Research

JoVE Journal

Biology

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

科研

生物学

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Crystallization of Membrane Proteins in Lipidic Mesophases

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

视黄醇运输的血浆视黄醇结合蛋白的膜受体的实时分析

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

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

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 ...

Imaging Plasma Membrane Deformations With pTIRFM

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

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 ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis MLKL 介导的血浆膜破裂特征

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...