这项研究是由赠款全球前沿项目（2011-0031944）和KRIBB研究计划项目的智能合成生物学中心的支持。

1.生物传感器的制备<ol><li>按照先前建立的协议2 CFP-MBP-YFP-的His6 -构建质粒pET21a（+）。 </li><li>接种5毫升的Luria肉汤（LB）的与大肠杆菌 DE3菌株的单个菌落，并在37°C孵育16小时振荡。 </li><li>传输1毫升的O / N培养物在含有100ml LB和在振荡培养箱中于37℃孵育直到在600nm（OD 600）的光密度达到0.5（约3小时）的500ml烧瓶中。 </li><li>在1,000×g下收获在50ml的锥形管中的细胞通过离心在4℃下20分钟。 </li><li>用50毫升冰冷的蒸馏水（DW）和离心机快速重悬沉淀在每个管中，在1000×g下在4℃下20分钟。 </li><li>通过轻轻摇动直到溶液（电感受态细胞），用10％（V / V）甘油悬浮在50μl冰冷的DW粒料达到的OD 600 </suB&gt; 100。 </li><li>放置电感受态细胞的混合物（50微升细胞以OD的100 600）和质粒pET21a（+），10纳克- CFP-MBP-YFP-的His6在冰冷电穿孔管在电穿孔设备和电穿孔该混合物（18千伏/厘米，25μF）。 </li><li>迅速1毫升SOC培养基添加到反应杯和重悬轻轻细胞，然后回收在37℃下1小时，在15毫升的圆底管轻轻摇动。 </li><li>涂布在含有100微克/毫升氨苄青霉素的LB板中的细胞，并在37℃孵育12小时。 </li><li>使用循环隔离单菌落并接种菌落在10ml含有LB，在37℃100微克/ ml氨苄青霉素的摇动12小时的。 </li><li>将5ml种子培养添加到500毫升的LB含有100μg/ ml氨苄青霉素和孵育培养在37℃振荡培养箱中。 </li><li>添加0.5mM的异丙基β-D-硫代半乳糖苷（IPTG）的外径600到达时0.5孵育在37℃振荡培养箱中培养24小时。 </li><li>离心机的细胞在4500×g的20分钟（4℃），并轻轻取出上清。 </li><li>重新悬浮于5mL的结合缓冲液（20mM的Tris-HCl，pH值8.0，1mM的PMSF，0.5mM的EDTA，和1mM DTT）沉淀。 </li><li>超声处理上有六个10秒连发冰细胞在200-300瓦，下面的每个突发冷却10秒。 </li><li>离心裂解物在10,000× 克30分钟，在4℃以沉淀细胞碎片。转移上清液（可溶性蛋白）到一个新的收集管。 </li><li>为了实现FRET传感器蛋白质的亲和纯化，负载4毫升澄清的细胞裂解物上样到Ni-NTA亲和柱（5毫升体积），用快速蛋白液相层析（FPLC）18执行色谱测定。 </li><li>用洗涤缓冲液五个柱体积洗一次列I（50mM磷酸盐缓冲液，300毫氯化钠，10mM咪唑，pH 7.0）中。 </li><li>重复该洗涤步骤用洗涤缓冲液II五个柱体积（50mM磷酸盐缓冲液，300毫氯化钠，的20mM咪唑，pH 7.0）中。 </li><li>用洗脱缓冲液五个柱体积（50mM磷酸盐缓冲液，300毫氯化钠，500mM咪唑，pH 7.0）中洗脱传感器蛋白。 </li><li>浓缩和脱盐洗脱样品，填充高达20样品和离心机毫升浓缩器（10,000兆瓦膜尺寸），用于在3000×g下10分钟。重新填充用0.8％的磷酸盐缓冲盐水（PBS）浓缩。重复此步骤两次，第一次填充聚光用20ml样品，然后用PBS再填充。 </li><li>回收浓缩和脱盐传感器蛋白并将其储存在-80℃。 </li></ol> 2.使用FRET分析糖含量的测定注:FRET分析仪结构的细节在我们以前的工作7进行了描述。 <ol><li>制备含0.2 0.8％PBS的检测解决方案传感器蛋白质微米。 </li><li>打开FRET分析仪。按"向上"按钮2秒来校准最佳温度。用"UP"和"DOWN"按钮，将温度设置为53℃，并按下"SET"键。 </li><li>对于校准，请同时按住"UP"和"DOWN"按钮2秒。确认LED面板显示"CALIB"，然后按"SET"键。 </li><li>将只含PBS缓冲液进入分析仪的试管架，然后按"SET"键12.5×12.5×45毫米（长×宽×高）的长方体容器（试管）。 </li><li>以仅包含检测溶液（见2.1）不加糖（麦芽糖/蔗糖）更换试管，然后按"SET"键来校准基准。 </li><li>更换含10毫米糖检测解决方案，一个比色皿，并按下"SET"按钮。 </li><li>以确定一个饮料样品中的糖含量，把1毫升的饮料样品中的1.5毫升微量离心管中并离心16,000×g的1分钟。 注意:基于传感器FRET荧光测定具有不需要特殊的预处理样品的样品，因为只有1％（V / V）的优点包括在总体积。但是，我们建议去除可能影响荧光测定（ 例如，细胞，不溶性微粒，脂质，脂肪，或具有自发荧光的任何材料）的任何材料。此外，如果一强酸，强碱，清洁剂（洗涤剂），或乳化剂（乳化剂）的存在以高浓度并可能影响FRET生物传感器的特性，应当使用有机溶剂或除去中和剂。例如，当乳品脂肪和乳化剂选自冷冻点心消除，将样品以16,000×g离心以离心管30分钟，液体between中的底部沉积物和乳品脂肪顶层被提取。然后己烷等量加入，然后离心以15,000×g的30分钟，以消除脂质。 </li><li>用1-ml注射器除去上清液，并通过注射器过滤器（孔径为0.2μm）进行过滤。 </li><li>放置0.1毫升过滤饮料样品中含有0.9毫升PBS中，并涡旋轻轻1.5毫升离心管中。 注:这是正确稀释饮料样品的关键。在这种情况下，进行1000倍稀释，以使糖浓度将落在该装置的动态范围内。我们建议通过参照在饮料的标签的糖含量预先估计目标糖浓度。 </li><li>加入5微升稀释饮料样品（1％，V / V）的含有0.495毫升检测溶液的反应杯中。 </li><li>放置反应杯在FRET分析仪的反应杯保持器和预热样品溶液至53℃。 </li><li>按"SET"键来测量糖含量。 注意可以通过在五百三十五分之四百八十八毫微米7,8-读取比率评价使用配备有用于温度控制的珀尔帖装置的多标记平板读数器或荧光分光光度计FRET的测量。对于蔗糖的检测，请按照1.1的步骤2.12与CSY-LH传感器2。 </li></ol>

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作者什么都没有透露。

该协议允许饮料样品中的糖含量的快速和有效的量化，使用定做的FRET分析器7在用于FRET传感器的最佳温度。分析器被设计用最近开发的，廉价的405纳米波段紫外LED作为光源和两个光检测器与硅光电二极管。这个装置是更符合成本效益比其它可比的荧光计。该器件显示高检测灵敏度，在最佳的温度范围为FRET传感器测量两个发射波长带（530纳米和480纳米）的比率特别时。它的灵敏度和强度检?...

荧光共振能量转移（FRET）已被广泛地用作生物统计传感器来检测小分子如糖，钙离子，氨基酸1-4。 FRET生物传感器含有荧光蛋白，青色荧光蛋白（国家重点计划）和黄色荧光蛋白（YFPs），其稠合到周质结合蛋白（的PBPs）的两端。糖类结合到位于FRET传感器的中间的PBPs，从而导致其随后改变在的PBPs的一端的两个荧光蛋白的距离和跃迁偶极取向传感器的结构变化。这种改变，通过测量EYFP（530纳米），ECFP（480纳米）的发光波长的比率使得能够糖含量的定量分析。由于高灵敏度，特异性，实时监测能力，和FRET生物传感器的响应时间快，这些传感器被广泛应用于环境，工业和医疗应用5。此外，比率式使用FRET生物传感器etric测量具有重要的实际的好处，因为它可以用来测量复杂生物样品，其中所述传感器的浓度不能被容易地控制和背景荧光总是存在于组件。 尽管基于FRET的传感器，用于定量可视化的这些优点，不完整的域名运动转移到荧光蛋白微小的结构性变化产生固有的弱信号强度。这个弱信号限制基于FRET的传感器，用于在体外或体内分析6的应用程序。因此，大多数FRET生物传感器需要使用昂贵的，高度敏感的设备。先前，我们开发了一种廉价和便携式FRET分析仪类似于现有荧光分析仪7的功能。在该装置中，廉价的405纳米波段紫外发光二极管（LED）作为光源，使第激发Ë荧光信号，更换昂贵的灯泡或激光。分析仪的检测系统的散热荧光信号高效率地聚焦到两个光电检测器与硅光电二极管。在最近的研究中，我们发现，在50检测温度的优化- 55°C可能显著放大比例FRET信号8。这个特定的温度信号增强，与定做的FRET分析仪一起使得能够快速且高灵敏度更一般诊断应用中的使用的FRET传感器。 在这个协议中，我们通过量化市售饮料的糖含量表现出最佳的FRET的温度条件下的FRET分析仪的通用性。这个协议提供了FRET设备操作的细节，以及传感器和样品制备的简要说明。我们预计，该报告将推动便携式的潜在应用分析器在小规模的实验室环境，并提供与基于FRET的生物传感器的廉价的现场诊断装置的进一步发展奠定了基础。

<table><tbody><tr><td>LB</td><td>BD</td><td>#244620</td><td></td></tr><tr><td>isopropyl &amp;beta;-D-thiogalactoside (IPTG)</td><td>Sigma</td><td>I6758</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9518</td><td></td></tr><tr><td>Tri-HCl</td><td>Bioneer</td><td>C-9006-1</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>78830</td><td></td></tr><tr><td>EDTA</td><td>Bioneer</td><td>C-9007</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D0632</td><td></td></tr><tr><td>NaCl</td><td>Junsei</td><td>19015-0350</td><td></td></tr><tr><td>phosphate-buffered saline (PBS)</td><td>Gibco</td><td>70011-044</td><td>0.8% NaCl, 0.02% KCl, 0.0144% Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.024% KH&lt;sub&gt;2&lt;/sub&gt;OP&lt;sub&gt;4, &lt;/sub&gt;pH 7.4</td></tr><tr><td>SOC</td><td></td><td></td><td>2% tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MGCl&lt;sub&gt;2&lt;/sub&gt;, 20 mM Glucose</td></tr><tr><td>Resource Q</td><td>Amersham Biosciences</td><td>17-1177-01</td><td>6 &amp;times; 30 mm anion-exchange chromatography column&amp;nbsp;</td></tr><tr><td>HisTrap HP1</td><td>Amersham Biosciences</td><td>29-0510-21</td><td></td></tr><tr><td>Quartz cuvette</td><td>Sigma</td><td>Z802875</td><td></td></tr><tr><td>AK&amp;Auml;KTAFPLC</td><td>Amersham Biosciences</td><td>18-1900-26</td><td>a fast protein liquid chromatography (FPLC)</td></tr><tr><td>Cary Eclipse</td><td>VarianInc</td><td></td><td>a fluorescence spectrophotometer</td></tr><tr><td>VICTOR&amp;nbsp;&amp;nbsp;</td><td>PerkinElmer</td><td>2030-0050</td><td>a multilabel plate reader</td></tr><tr><td>&lt;i&gt;E. coli&lt;/i&gt; JM109 (DE3)</td><td>Promega</td><td></td><td>Electrocompetent cells</td></tr><tr><td>A (Beverage)</td><td>Korea Yakult Co. (Korea)</td><td>Birak</td><td>Fermented drinks</td></tr><tr><td>B (Beverage)</td><td>Lotte Foods (Korea)</td><td>Epro</td><td>Soft drink</td></tr><tr><td>C (Beverage)</td><td>Lotte Foods (Korea)</td><td>Getoray</td><td>Sports drink</td></tr></tbody></table>

highly sensitive and rapid fluorescence detection with a portable fret analyzer

在荧光共振能量转移最近的改进（FRET）传感器已经使它们的使用，以检测各种小分子，包括离子和氨基酸。然而，FRET传感器的先天弱信号强度，以防止在各个领域的应用并使得使用昂贵的，高端的荧光计必要的一个重大挑战。先前，我们构建可以特异性测量两个发射波长带（530和480纳米）的比率，以实现高的检测灵敏度具有成本效益的，高性能的FRET分析仪。最近，人们发现FRET传感器与细菌周质结合蛋白检测用在临界温度范围的50最高灵敏度的配体 - 55℃。这份报告描述了使用我们的便携式FRET分析仪具有特定的温度-FRET传感器市售饮料样品评估中含糖量的协议。我们的结果表明，附加的预热FRET的传感器的过程显著增加的FRET比率信号，以使糖含量更精确的测量。定做的FRET分析仪和传感器成功地应用到量化糖含量在三种类型的商业饮料。我们预计，设备的尺寸进一步降低和性能的提高将有利于环境中使用手持式分析仪，其中高端装备不可用。

以执行使用FRET分析糖含量的定量分析，必须建立一个拟合曲线推定从观察的FRET比率目标糖浓度。设r定义CFP的发光强度的比在480nm和在530nm（式1）中产生的YFP的发光强度。 <img alt="式（1）" src="/files/ftp_upload/54144/54144equation1.jpg" />可以通过观察FRET比r，在不同的糖浓度来?...

Watch this Scientific Journal Video about Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer at JoVE.com

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

这个协议描述了使用定制便携式FRET分析器荧光共振能量转移（FRET）的传感器数据的快速且高灵敏度定量。该装置被用于最大化的检测灵敏度，有利的糖含量的实际和有效的评估的临界温度范围内检测麦芽糖。

高度敏感和快速荧光检测用便携式分析仪FRET

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

highly-sensitive-rapid-fluorescence-detection-with-portable-fret

Research

JoVE Journal

Biochemistry

9.0K 次观看.  Korea Research Institute of Bioscience and Biotechnology. 这个协议描述了使用定制便携式FRET分析器荧光共振能量转移（FRET）的传感器数据的快速且高灵敏度定量。该装置被用于最大化的检测灵敏度，有利的糖含量的实际和有效的评估的临界温度范围内检测麦芽糖。 

视频: Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

科研

生物学

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

荧光技术结合测定活细胞中的力敏感蛋白动力学

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

生物工程

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

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

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

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 ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

移动式单分子FRET探头的自动化二维时空分析

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

通过电穿孔内在和生物分子荧光观察生活中的微生物

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

开源高内涵分析利用自动荧光寿命成像显微镜

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded F&#246;rster&#8217;s Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis.

使用活细胞中的构象FRET生物传感器实时监测极光激酶A活化

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

评估具有FRET敏化发射的活细胞中的蛋白质相互作用

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...