Name: live cell imaging of f-actin dynamics via fluorescent speckle microscopy (fsm)
Uploaded: 2009-08-05T19:00:00
Description: Watch this Scientific Journal Video about Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM at JoVE.com

由美国国立卫生研究院授予U01 GM06230 qFSM发展。

第1部分：获取荧光标记的肌动蛋白为密克罗尼西亚所需材料：纯化的肌动蛋白，荧光（Alexa的，X -罗丹明建议）。 G缓冲区，超速离心机<ol><li>取得良好的密克罗尼西亚电影的一个重要组成部分，需要肌动蛋白与您所选择的荧光团适当的标记（或其他细胞骨架蛋白）。 我们建议使用Alexa的荧光团（488，568波长）和X -罗丹明琥珀酰亚胺酯目标暴露在表面的肌动蛋白赖氨酸残...

<ol>
	<li>Waterman-Storer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+speckle+microscopy+(FSM)+of+microtubules+and+actin+in+living+cells.">Fluorescent speckle microscopy (FSM) of microtubules and actin in living cells.</a> Curr Protoc Cell Biol. Chapter 4, Unit 4.10-Unit 4.10 (2002).</li><li>Ponti, A., Machacek, M., Gupton, S. L., Waterman-Storer, C. M., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+actin+networks+drive+the+protrusion+of+migrating+cells.">Two distinct actin networks drive the protrusion of migrating cells.</a> Science. 305, 1782-1786 (2004).</li><li>Zhang, X. F., Schaefer, A. W., Burnette, D. T., Schoonderwoert, V. T., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-dependent+contractile+responses+in+the+neuronal+growth+cone+are+independent+of+classical+peripheral+retrograde+actin+flow.">Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow.</a> Neuron. 40, 931-944 (2003).</li><li>Salmon, W. C., Adams, M. C., Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-wavelength+fluorescent+speckle+microscopy+reveals+coupling+of+microtubule+and+actin+movements+in+migrating+cells.">Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells.</a> J Cell Biol. 158, 31-37 (2002).</li><li>Danuser, G., Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fluorescent+speckle+microscopy+of+cytoskeleton+dynamics.">Quantitative fluorescent speckle microscopy of cytoskeleton dynamics.</a> Annu Rev Biophys Biomol Struct. 35, 361-387 (2006).</li></ol>

几个关键因素是至关重要的，成功获得散斑图像，但是良好的斑点，开始和结束与荧光标记的肌动蛋白的质量。一个标签的比例高，肌动蛋白单体，约0.4至0.7的染料将确保斑点会显得明亮和离散，标记的蛋白质本身，而应可溶性聚集。同样重要的是显微注射过程。为了保证正常细胞动态平衡和生存，细胞需要被注入一个低流量的压力，引入低浓度的标记蛋白。这需要一定的技术专业知识/经验的显...

注射缓冲器（IB）， 50毫米钾谷氨酸 0.5 M的氯化镁（ 附录2A） 在-20 ° C储存长达2年 &nbsp; G缓冲区 2毫米的Tris⋅氯，pH值8.0（ 附录2A） 0.2毫米氯化钙（ 附录2A） 只是在使用前添加： 0.2毫米ATP（见100毫米配方） 0.5毫米2 - 巯基乙醇店1天4 ° C

live cell imaging of f-actin dynamics via fluorescent speckle microscopy (fsm)

在这个协议中，我们描述了使用荧光斑点显微镜（FSM）来捕获PtK1细胞的肌动蛋白动力学的高清晰度图像。密克罗尼西亚的一个独特的优势是它能够捕捉到活细胞内的流动和周转动力学的F -肌动蛋白网络（组装/拆卸）。这种技术是有用的，特别是在衍生F -肌动蛋白动力学的定量测量与计算机视觉软件（qFSM）配对。我们描述了选择，显微注射法和荧光的肌动蛋白探针在活细胞中的可视化。更重要的是，类似的程序，适用于可视化等macomolecular集会。密克罗尼西亚已经证明微管，中间丝和粘附复合物。

Watch this Scientific Journal Video about Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM at JoVE.com

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

的选择，显微注射，并成像荧光标记的F -肌动蛋白，通过荧光斑点显微镜（密克罗尼西亚）。

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 ...

Research

JoVE Journal

Biology

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

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

三维超F-微丝高分辨率显微镜通过干涉光敏定位显微镜（iPALM）

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

在成细胞分化和融合过程中利用活成像跟踪核

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

活细胞成像，以评估在斜接主轴扰动后元相定时和细胞命运的动态

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...