Name: nanotopology of cell adhesion upon variable-angle total internal reflection fluorescence microscopy (va-tirfm)
Uploaded: 2012-10-02T12:00:00
Description: Watch this Scientific Journal Video about Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM at JoVE.com

作者感谢的土地，巴登-符腾堡州和Europäische联盟的Europäischer全宗献给模具REGIONALE Entwicklung -资金ZAFH光子N，Bundesministerium献给教化和Forschung（BMBF）资助的研究经费。 1792C08和巴登 - 符腾堡州基金会有限公司为融资项目“Aurami”的。

1。播种和培养细胞<ol><li>种单个细胞， 例如 U373-MG或U-251-MG的成胶质细胞瘤细 ​​胞在一个典型的密度为100个细胞/ mm 2的载玻片上，并成长为4872小时，在培养基中（ 例如与10％胎牛血清的RPMI 1640培养基和抗生素）使用的培养箱，在37℃和5％CO 2的 。 </li><li>孵育细胞的膜标记物， 例如 6-十二烷酰基-2-二甲基氨基萘（laurdan）60分钟施加在8μM（培养液中）4，...

<ol>
	<li>Gingell, D., Todd, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+reflection+microscopy:+a+quantitative+theory+of+image+interpretation+and+its+application+to+cell-substratum+separation+measurement.">Interference reflection microscopy: a quantitative theory of image interpretation and its application to cell-substratum separation measurement.</a> Biophys. J. 26, 507-526 (1979).</li><li>Reichert, W. M., Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+reflection+fluorescence+(TIRF)+microscopy+(I)+Modelling+cell+contact+region+fluorescence.">Total internal reflection fluorescence (TIRF) microscopy (I) Modelling cell contact region fluorescence.</a> J. Cell Sci. 96, 219-230 (1990).</li><li>Stock, K., Sailer, R., Strauss, W. S. L., Lyttek, M., Steiner, R., Schneckenburger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable-angle+total+internal+reflection+fluorescence+microscopy+(VA-TIRFM):+realization+and+application+of+a+compact+illumination+device.">Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device.</a> J. Microsc. 211, 19-29 (2003).</li><li>Wagner, M., Weber, P., Strauss, W. S. L., Lassalle, H. -. P., Schneckenburger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotomography+of+cell+surfaces+with+evanescent+fields.">Nanotomography of cell surfaces with evanescent fields.</a> Adv. Opt. Technol. 2008, 254317 (2008).</li><li>Lassalle, H. -. P., Baumann, H., Strauss, W. S. L., Schneckenburger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-substrate+topology+upon+ALA-PDT+using+variable-angle+total+internal+reflection+fluorescence+microscopy+(VA-TIRFM).">Cell-substrate topology upon ALA-PDT using variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).</a> J. Environ. Pathol. Toxicol Oncol. 26, 83-88 (2007).</li><li>Coates, C. G., Denvir, D. J., McHale, N. G., Thornbury, K. D., Hollywood, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+low-light+microscopy+with+back-illuminated+electron+multiplying+charge-coupled+device:+enhanced+sensitivity,+speed+and+resolution.">Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed and resolution.</a> J. Biomed. Opt. 9, 1244-1252 (2004).</li><li>Gustafsson, M. G. L., Shao, L., Carlton, P. M., Wang, C. J. R., Golubovskaya, I. N., Cande, W. Z., Agard, D. A., Sedat, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+resolution+doubling+in+wide-field+fluorescence+microscopy+by+structured+illumination.">Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination.</a> Biophys. J. 94, 4957-4970 (2008).</li><li>Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W., Olenych, S., Bonifacino, J. S., Davidson, M. W., Lippincott-Schwartz, J., Hess, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313, 1642-1645 (2006).</li><li>Hess, S. T., Girirajan, T. P. K., Mason, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy.">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophys. J. 91, 4258-4272 (2006).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat. Meth. 3, 793-796 (2006).</li><li>Willig, K. I., Harke, B., Medda, R., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+microscopy+with+continuous+wave+beams.">STED microscopy with continuous wave beams.</a> Nat. Meth. 4, 915-918 (2007).</li><li>Schneckenburger, H., Wagner, M., Weber, P., Bruns, T., Richter, V., Strauss, W. S. L., Wittig, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Dimensional+Fluorescence+Microscopy+of+Living+Cells.">Multi-Dimensional Fluorescence Microscopy of Living Cells.</a> J. Biophotonics. 3, 143-149 (2011).</li><li>Dougherty, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosensitizers:+therapy+and+detection+of+malignant+tumours.">Photosensitizers: therapy and detection of malignant tumours.</a> Photochem. Photobiol. 45, 879-889 (1987).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-substrate+contacts+illuminated+by+total+internal+reflection+fluorescence.">Cell-substrate contacts illuminated by total internal reflection fluorescence.</a> J. Cell Biol. 89, 141-145 (1981).</li><li>&#214;lveczky, B. P., Periasamy, N., Verkman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+fluorophore+distribution+in+three+dimensions+by+quantitstive+multiple+angle+total+internal+reflection+fluorescence+microscopy.">Mapping fluorophore distribution in three dimensions by quantitstive multiple angle total internal reflection fluorescence microscopy.</a> Biophys. J. 73, 2836-2847 (1997).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+surface+fluorescence+with+vry+high+aperture+microscope+objectives.">Selective imaging of surface fluorescence with vry high aperture microscope objectives.</a> J. Biomed. Opt. 6, 6-13 (2001).</li><li>Schneckenburger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+reflection+microscopy:+technical+innovations+and+novel+applications.">Total internal reflection microscopy: technical innovations and novel applications.</a> Curr. Opin. Biotechnol. 16, 13-18 (2005).</li></ol>

描述了一种方法，用于测量细胞衬底与纳米精度的距离。目前，超高分辨率显微镜的方法， 例如 ，基于结构照明7或单分子检测8-10以及受激发射损耗（STED）显微镜是相当大的兴趣。然而，没有这些技术允许的轴向分辨率低于50nm。此外，需要相当高的为50100 W / cm 2的辐照度为单分子的方法和超过3000瓦/厘米2的受激发射损耗显微镜。这超过太阳辐照度（约100毫...

当光束传播通过的介质的折射率n 1，满足的第二介质的折射率n 2 &lt;n 1的一个接口，全内反射发生的所有角度，这是大于临界角的入射角ΘΘ=反正弦（N 2 / N 1）。尽管被全反射入射光束的唤起的瞬态电磁场，渗入所述第二介质和呈指数衰减，与从界面的垂直距离z根据I = I 0（z）的易通/天（Θ）。 I（z）的对应的电磁场和d（Θ）的强度，所给出的在波长λ的穿透深度 D（θ）=（λ/4π）（1 2罪2Θ - N 2 2）-1 / 2 据报道在文献1,2中 ，I（0） </sUB&gt; 站 |电子乘以传输系数T（Θ），比n 2 / n 1个与入射光的强度相对应。如果该光束的电场矢量的偏振垂直于入射平面的（ 即所跨越的入射和反射光束的平面），该传输系数由下式给出 T（Θ）= 4余弦2Θ/ [1 - （n 2个 / nL的1）2] 对于检测到的荧光强度在TIRFM测量中，光吸收具有集成的样品的所有层和乘以有关染料的荧光量子收率η，以及检测与立体角Ω。正如以前报道的3，这种强度可以通过以下进行说明 我F（Θ）= AT（Θ）OC（Z）E-Z / D（Θ）DZ 如果所有这些因素是独立的f<html>的ROM的入射角Θ和坐标z均包括在实验常数A.等式3可以解析解，如果荧光基团的浓度c（z）的被认为是几乎恒定的 -或者， 例如 ，在所有坐标Z≥Δ的细胞质内的细胞，从该接口具有的距离Δ。在这种情况下，积分计算从z =Δ到z =∞导致 I F = A C T（Θ）D（Θ）E-Δ/ D（Θ） -或之间的Z =Δ -吨/ 2和z =Δ+ t / 2只， 即一层薄薄的厚度t内， 例如位于从界面的距离Δ的细胞膜。在这种情况下，可以计算出的荧光强度为 I F =是C T（Θ）TE-Δ/ D（Θ） 如果tΔ比较小。 耳鼻喉科“&gt;从方程（4）和（5）细胞-基底距离Δ，如果可以计算出的荧光强度I F的图像被记录为可变角度Θ，如果In（I F / T d的）（公式4）或ln（I F / T）（公式5）被评价为1次/ d的函数的这些角度，对于本文中所用的膜标记laurdan（第下文）根据公式5进行评价。 正如以前报道的3，图像采集和评估要求 - 在样本激发光的均匀分布的， -有效性的2层模型（与折光率n 1和n 2分别为在基板和细胞质，分别）。这认为，如果等离子体膜是非常薄的，如果在细胞外介质中的层（细胞和衬底之间）与入射光的波长相比是很小的。 此外，温和的数值孔径（通常为A≤0.90）的显微镜物镜是有利的，以避免由于辐射的各向异性，在近场中的电介质界面亮度偏差。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 类型 </td><td> 评论 </td></tr><tr><td>恒温箱</td><td> Nunc公司</td><td> Nunc公司蜂星 QM1300SVBA </td><td></td></tr><tr><td>层流</td><td>霍尔滕安全</td><td> S2010 1.2 EN GG 1LN </td><td></td></tr><tr><td> DMEM +10％FCS + 1％青霉素/链霉素</td><td> BIOCHROM </td><td></td><td>栽培介质</td></tr><tr><td>玻璃对象的幻灯片</td><td>马林费尔德</td><td>纯白色的玻璃</td><td>使用了特殊的清洗程序</td></tr><tr><td> 6-十二烷酰基-2-二甲基氨基萘（laurdan） </td><td>分子探针</td><td></td><td>荧光标记（储备液：2 mm的乙醇） </td></tr><tr><td>显微镜</td><td>卡尔·蔡司</td><td> Axioplan 1 </tD&gt; <td></td></tr><tr><td>激光二极管</td><td> PicoQuant </td><td> LDH 400与驱动程序PDL 800-B </td><td>波长：391纳米</td></tr><tr><td>单模光纤系统</td><td>点源</td><td> kineFlex </td><td>可用于准直光学器件</td></tr><tr><td> EMCCD相机</td><td>安道​​尔</td><td> DV887DC </td><td>背照式摄像头</td></tr></tbody></table>

nanotopology of cell adhesion upon variable-angle total internal reflection fluorescence microscopy (va-tirfm)

表面的拓扑结构， 例如，在衬底上生长的细胞中，确定由可变角度总内部反射荧光显微镜（VA-TIRFM）与纳米精度。将细胞培养在透明的幻灯片，并与荧光标记物均匀地分布在它们的细胞膜中孵育。照明发生由一个平行的激光束根据变量的总内部反射（TIR）的角度，用不同的穿透深度的瞬态电磁场。荧光图像后，在约10个不同的角度照射的记录允许，计算细胞衬底的距离的精度为几纳米。这样确定的各种细胞株， 例如，肿瘤细胞和恶性细胞少，密合性的差异。此外，可能发生的变化时的细胞粘附化学或光动力治疗可以进行检查。与其他方法相比，超分辨率显微镜的光照射保持活细胞的非常小，并且没有损坏预期发生。

Watch this Scientific Journal Video about Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM at JoVE.com

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

细胞粘附在基板上的拓扑结构是由可变角度的总内部反射荧光显微镜（VA-TIRFM）与纳米精度测量。

Nanotopology细胞粘附在可变角度的全内反射荧光显微镜（VA-TIRFM）

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence ...

Research

JoVE Journal

Bioengineering

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

科研

生物工程

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

珠聚集测定假定的细胞粘附分子的表征

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

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

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 ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

光敏定位显微镜与双分子荧光互补（附设-PALM）

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...