Name: fret microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors
Uploaded: 2012-08-20T13:00:00
Description: Watch this Scientific Journal Video about FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors at JoVE.com

作者要感谢安科Rüttgeroth和卡琳娜齐默尔曼的技术援助。这项工作得到了德意志研究联合会（授予NI 1301/1-1 VON）和德国哥廷根大学医学中心（“富利”授予VON）。

1。建立一个共振成像显微镜原则上，任何倒置荧光显微镜，这是在实验中，有一个摄像头端口，可以适用于FRET成像。最后的安装应包括下列关键组成部分：一台显微镜，一个光源或无需额外的快门，以及一个CCD摄像头（参见图1）发射光的光束分离器，用于。的硬件设备，特别是光源，快门和相机被集成到由成像的图像采集和分析的软件，该软件允许控制。下面?...

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在该协议中，我们演示了如何构建一个简单的低成本但功能强大的共振成像系统可用的生物传感器具有多种常规应用。这里介绍的系统被设计为CFP和YFP，或类似类型的荧光蛋白，作为供体 - 受体对。与此同时，以及其他个人的生物传感器成为可用的，使用例如绿色和红色荧光蛋白14。为了适应所描述的系统为其他颜色，应选择适当的光源和/或过滤套。在LED，另一个单个LED的行的情况下，?...

<table border="1"><tbody><tr><td> 的试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> BES缓冲级</td><td> AppliChem </td><td> A1062 </td><td></td></tr><tr><td> 氯化钙二水</td><td> Sigma-Aldrich公司</td><td> C5010 </td><td></td></tr><tr><td>玻璃coverslides </td><td> Thermo Scientific的</td><td> 004710781 </td><td>直径24毫米</td></tr><tr><td>玻璃底细胞培养皿</td><td>世界精密仪器</td><td> FD3510-100 </td><td></td></tr><tr><td> D-MEM培养基</td><td> Biochrom AG </td><td> F0445 </td><td></td></tr><tr><td>胎儿小牛血清（FCS） </td><td> Thermo Scientific的</td><td> SH30073.02 </td><td></td></tr><tr><td> L-谷氨酰胺</td><td> Biochrom AG </td><td> K0283 </td><td></td></tr><tr><td> HEPES </td><td>西格玛</td><td> H4034 </td><td></td></tr><tr><td>氯化钾</td><td>西格玛</td><td> P5405 </td><td></td></tr><tr><td> 氯化镁六水</td><td> AppliChem </td><td> A4425 </td><td></td></tr><tr><td>氯化钠</td><td> AppliChem </td><td> A1149 </td><td></td></tr><tr><td>的Na 2 HPO 4 </td><td> Sigma-Aldrich公司</td><td> S9707 </td><td></td></tr><tr><td>青霉素/链霉素</td><td> Biochrom AG </td><td> A2213 </td><td></td></tr><tr><td>倒置荧光显微镜</td><td>例如，尼康</td><td>尼康请求</td><td></td></tr><tr><td> CoolLED </td><td> CoolLED </td><td> P E-100 </td><td> 440 nm处</td></tr><tr><td>双屏显示</td><td>光度</tD&gt; <td> DV2-SYS </td><td></td></tr><tr><td> DualView的过滤器滑块</td><td>光度</td><td> 05-EM </td><td></td></tr><tr><td> CFP / YFP滤波器组的</td><td>色度技术</td><td> 49052 </td><td>未经发射滤波器</td></tr><tr><td> ORCA-03G摄像头</td><td>日本滨松光子学</td><td> C8484-03G02 </td><td></td></tr><tr><td> Arduino的I / O板</td><td> Sparkfun电子</td><td> DEV-00666 </td><td></td></tr><tr><td> Attofluor细胞室</td><td> Invitrogen公司</td><td> A-7816 </td><td></td></tr><tr><td> WindowsXP或Windows7系统的个人电脑</td><td>任何供应商</td><td></td><td>包括硬盘驱动器的高容量</td></tr></tbody></table>

fret microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors

福斯特共振能量转移（FRET）显微镜将继续作为一个在活细胞和组织的生化事件和信号的实时监控技术获得越来越多的关注。与传统的生化方法相比，这种新技术的特点是高时空分辨率的。 FRET实验使用不同的基因编码的生物传感器，它可以表示随着时间的推移， 在现场或在体内 1-2和成像。典型的生物传感器可以报告通过测量FRET的荧光团标记的对蛋白质或构象的变化，在一个单一的蛋白质窝藏景点3-4的分子与结合部分相互连接的供体和受体荧光团之间的蛋白质-蛋白质相互作用。蛋白质-蛋白质相互作用的双分子生物传感器包括，例如，构建设计用于监控电池单元5中的G-蛋白激活，而单 ​​分子传感器测量被广泛地用于图像的第二信使如钙6中，cAMP 7-8，肌醇磷酸盐9和cGMP 10-11一定的构象变化。在这里，我们将介绍如何建立一个定制的萤光共振成像系统，从单一的商业可用的组件，如何控制整个安装使用微管理器免费。这个简单但功能强大的仪器是专为在活细胞中的常规或更复杂的FRET测量。自写插件使用过程中的变化在FRET比在实时可视化图形格式被存储在任何实验之前获得的图像处理兼容内置的ImageJ的免费软件，用于后续的数据分析。这种低成本系统的特点是灵活性高，可以成功地用于监测各种的生化事件和信号分子提供了大量的共振生物传感器在活细胞和组织。作为一个例子，我们展示了ĤOW使用这种成像系统进行实时监控中cAMP现场293A细胞刺激后，与β-肾上腺素能受体激动剂和阻滞剂。

Watch this Scientific Journal Video about FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors at JoVE.com

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

福斯特共振能量转移（FRET）显微镜是一种功能强大的技术，使用各种生物传感器记者在活细胞中的信号转导事件的实时监控。在这里，我们将介绍如何建立一个自定义的表面荧光共振成像系统从商业上可用的组件和如何使用它的FRET实验。

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Research

JoVE Journal

Biology

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

科研

生物学

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

实时监控的配体 - 受体相互作用的荧光共振能量转移

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

在Live哺乳动物细胞中质粒复制多代荧光显微镜监测

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

的细胞内信号与相邻小区发生细胞凋亡通过互动活细胞诱导活动标识

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

飞秒激光的光刺激激活目标细胞中的细胞外信号调节激酶(ERK)信号或线粒体事件

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...