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。下面我们将介绍一个过程来组装一个简单的共振系统，从商业上可用的组件。 <ol><li>将光源的显微镜。例如，使用单波长的发光二极管（CoolLED P E-100，440 nm处），选择性增强型青色荧光蛋白（CFP）作为捐助者中最兴奋的共振生物传感器。它可以直接ly和容易地连接到落射荧光显微镜的照明端口。还有也多波长的LED可用，可以以类似的方式连接。相反，可以使用LED，以及其他如XBO75氙弧灯（通常用于奥林巴斯和蔡司显微镜）或HBO汞灯（通常尼康显微镜上安装）标准光源。在荧光灯的情况下，你也应该放置在灯和照明端口启用辅助软件到您的样品的激发光来控制快门。 CoolLED不要求任何额外的快门，因为它可以直接接通和断开由软件。单色LED系统的缺点是有限数量的激发波长，而与各过滤器组的荧光灯是一种更加灵活的选择，特别是当使用多个和双分子的生物传感器。另外，单色光源（ 如多彩V，TILLPhotonics），他们通常有一个集成的快门，可以通过软件控制触发信号。 </li><li>将适当的过滤器多维数据集在显微镜。对于常规的FRET测量CFP和增强型黄色荧光蛋白（YFP）或它们的变体作为FRET对，我们用一个简单的过滤器立方体包含的ET436/30M激发过滤器（使用LED时，可以省略，但不可缺少的当使用了氙气或汞灯，而不是LED）和一个DCLP455分色镜。显微镜时，应配备适合一个很好的高分辨率荧光显微镜的目标，例如一个计划，氟石，计划NEOFLUAR或计划复消色差透镜40倍，60倍或100倍油浸目标。 </li><li>上的荧光的光开关，并检查是否被均匀地分布在整个视场的光点。如果不是这种情况下，附加的对准的LED或灯泡所需的检体，以达到最佳的照明。这可以是PERFORMED通过使用在空间定位的二极管或灯泡的螺钉。 </li><li>连接通过一个C型的光束分离器一个显微镜的排放端口。例如，使用DV2双屏显示（配光曲线），这将发射光分为两个通道（供体和受体）在一个单一的CCD摄像头芯片可以同时监测。另外，还有一些其他可比较的产品，如Optosplit（大老山研究）或滨松ORCA-D2的双波长的摄像头集成到一个分束器。对于CFP / YFP FRET对，我们使用05-EM过滤器集，其中包含的505dcxr分色镜加ET48​​0/30M和ET535/40M排放过滤器，CFP和YFP，分别提供与DV2。取而代之的是分束器，两个滤波器立方体为供体（含有ET436/30M激发CFP，DCLP455分色镜和CFP发射滤光器的滤波器，用于）和受体（含有的CFP激发滤光器，DCLP455和YFP的发射滤光片）信道被用于在许多共振系统。另外，一个过滤器，可以安装在镜头前没有任何排放过滤器和自动发射滤光片轮位置的立方体。在这种情况下，电动显微镜或两个发射过滤器的位置在〜200-300毫秒内的滤光轮之间交替执行比例式成像。这种轻微的延迟是可以接受的，成像时相当慢的细胞内过程，如cAMP信号，其中真正的同时采集两个通道是不是关键的。 </li><li>连接的CCD摄像机（使用例如ORCA-03G或从Hamamatsu Photonics ORCA-R2）的分束器。使用FireWire电缆将相机连接到计算机接口的IEEE1394，随相机附送的使用手册中的说明。在相机上安装摄像头驱动程序，而无需切换。 </li><li>最后，建立计算机和光源之间的通信，连接Arduino的I / O板（例如阿尔杜伊诺Duemilanove或Arduino的乌诺）的LED或t使用BNC连接线，它应该包含一个正常的BNC插头上的LED侧和两个单导线的另一端上，如在图2中示出的引脚GND（0）和8的电路板连接到o的快门。组装电路板，可直接连接到您的计算机的USB端口。 </li></ol> 2。设置的成像软件来控制和同步的光源与由相机捕获的图像，图像处理软件应安装在计算机上。有几种商业套装软件包括MetaFluor（分子式设备），Slidebook（智能成像创新），VisiView（Visitron系统）。在这里，我们演示了如何使用的开源微管理器的免费软件，它提供了高度的灵活性，低成本成像。 <ol><li>下载该软件的<a href="http://valelab.ucsf.edu/~MM/MMwiki/index.php/Micro-Manager_Version_Archive">http://valelab.ucsf.edu/〜的MM / MMwiki /指数。</a>PHP /微Manager_Version_Archive的。我们建议安装1.4.5发布，这是很容易配置在我们的手中。 </li><li>将Arduino板到您的计算机的USB端口。下载软件来控制Arduino板<a href="http://www.arduino.cc/en/Main/software" target="_blank">http://www.arduino.cc/en/Main/software</a> 。按照说明这个网站上找到并运行该软件只是一个时间开始之前，微经理。上传板微Manager软件使用的代码。该代码可以下载，在<a href="http://valelab.ucsf.edu/~MM/MMwiki/index.php/Arduino" target="_blank">http://valelab.ucsf.edu/〜的MM / MMwiki /的index.php / Arduino的</a> 。 </li><li>上的LED和摄像头切换。开始的微Manager之间的通信软件和配置的摄像头和LED（光源和快门）选择“工具”&gt;“硬件配置向导（ 见图3）。添加所需的组件，包括您的相机（ 即Hamamatsu_ DCAM）和，Arduino板（添加Arduino的开关，Arduino的轮毂和Arduino的快门装置）。在接下来的步骤中，向导建议使用默认设置。按向导提示时，保存新的系统配置。 </li><li>使用主菜单中打开“工具”&gt;“设备属性浏览器。向下滚动到“Arduino的开关状态”，选择“1”。关闭对话框。确保在主要的软件“对话框中勾选”自动快门“框。按“文件”&gt;“保存系统状态，应随时启动该软件后，打开保存的软件配置。这将建立董事会之间的沟通和所需要的软件进行图像采集。 </li><li>按“实时”监控来自摄像机的信号。确保在任何时间，“现场”或“快照”功能，选择荧光灯。在开始的第一次测量，按照说明您的光束分离器，每形成两个通道的光学对准。 </li></ol> 3。细胞培养和转染<ol><li>准备与蒸压轮24毫米玻璃coverslides（1 coverslide每孔）的6孔培养板。或者，玻璃有底的细胞培养皿中都可以使用。 </li><li>铠甲，293A细胞到板或菜在D-MEM培养基（补充了10％FCS，1％L-谷氨酰胺和1％青霉素/链霉素溶液），使细胞达到50-70％汇合后1天。 </li><li> 24小时后，电镀，转染的细胞在层流工作台与FRET传感器质粒用磷酸钙转染法（见3.5）。环磷酸腺苷生物传感器的质粒可以从本集团根据要求提供。读者也被称为7,8描述其他可用的环磷酸腺苷生物传感器的综合评论。 </li><li>在转染细胞，用于第一时间前，准备转染试剂。化妆的2.5M的CaCl 2和2xBBS解决方案（后者含有1.5毫摩尔的Na 2 HPO 4，50mM的BES，280 mM氯化钠，用NaOH调整到PH值为6.95），在去离子水中。无菌滤液的解决方案，使用常规的0.2微米的过滤器。 </li><li>要转染6孔板或6玻璃底船菜肴，混合10μg的FRET传感器质粒，加入50μl2.5M CaCl 2溶液和无菌水至500微升。拌匀。 </li><li>加入500μl的2个论坛。充分混匀，孵育10分钟，在室温下的混合物。 </li><li>吸取165μL转染混合溶液到每口井或菜。轻轻地搅动板，并把它放回孵化器。 FRET测量转染24小时后，细胞通常都愿意。确保细胞没有达到会合在这一点上，因为这可能影响几个细胞表面受体的活性。 </li></ol> 4。在活细胞中的荧光共振能量转移测量<ol><li>在第一时间测量之前，准备FRET的缓冲液含有144毫摩尔NaCl，5.4 mM KCl中，1毫摩尔MgCl 2，2mM的的CaCl 2，10 mM HEPES的在去离子水中，并用NaOH将pH调节至7.3。稀释成像实验与FRET缓冲器中要使用的化合物。 </li><li>启动图像处理软件。加载先前定义系统配置。选择“文件”&gt;“加载系统状态选择先前配置的系统状态。 </li><li>安装一个coverslide贴壁转染293A细胞成像室（ 如 Attofluor细胞室）。洗涤细胞，用荧光共振能量转移缓冲液和荧光共振能量转移缓冲液中加入400μl。当使用玻璃有底细胞培养皿洗涤贴壁细胞和缓冲每个培养皿中加入2毫升。我们执行所有测量FRET的缓冲液HEPES，所以，在室温下在无CO 2的控制是必要的。 </li><li>把一些浸油到目标转移到显微镜的成像室。专注于细胞层采用透射光。 </li><li>上的荧光韧带切换HT按“实时”按钮，选择一个单元格的实验。选择一个单元格与的最佳传感器表达，这意味着它应该不是太亮，不是太暗。找到一个合适的细胞后，立即关掉日光灯，以避免漂白FRET传感器。 </li><li>在“相机设置”的方式，导致一个很好的信号与噪声的比例，按“对齐”按钮后，所获取的图像（通常是10至50毫秒）调整曝光时间。 ，激发时间太长，可能会导致在漂白，过于短的时间内产生的图像质量低。 </li><li>按“多-D ACQ”。按钮和设置的时间点，并进行图像采集的时间间隔的数目。我们的营地-FRET传感器，我们获得了一个形象，每5秒。 </li><li>按“采集”按钮开始测量。 </li><li>在任何测量过程中，可以使用“FRET在线”插件（可在网上补充 ，所有的插件必须是缔约方会议IED微Manager软件的“插件”文件夹，然后再启动它）来监视FRET比例的变化。运行此插件，并在FRET比图像使用“写意选择”工具选择一个区域的利益。加入区域的ROI经理，按“平均”按钮，在“时间序列分析”窗口。 FRET比跟踪将被显示。在测量过程中，要更新跟踪运行的“FRETratioOnline2”插件，按“GetAverage”按钮。 </li><li>只要FRET的比例已经达到稳定的基线适用通过准确地移液入菜/腔室所需的化合物。具有药理化合物处理细胞，而不是简单的移液灌注系统可以使用在这个阶段。 </li><li>在实验完成后，保存时间的推移堆栈的图像。删除从显微镜和测量室，清洁使用目标组织的目标。 </li><li>返回到步骤4.3重复测量与新的样本。 </li></ol> 5。离线数据分析共振成像数据可以进行离线分析实验后，在任何时候使用ImageJ软件。作为此协议的补充，我们提供的的“FREToffline”插件，在我们的实验室获得的图像分割成供体和受体通道，并测量荧光强度在多个地区的利益。这些强度可以进一步复制粘贴到Excel或地电子表格，计算修正后的FRET比率。可视化的FRET单分子生物传感器的变化，简单谱仪经常被使用。在这种情况下，唯一的供体荧光团（CFP），被激发，并且采取两个图像在CFP和YFP的发射峰。计算出的比率YFP / CFP（有时也简称为FRET / CFP比）表示的两个荧光团之间的FRET的程度。在单分子的生物传感器，CFP和YFP部分的数目是相等的，因此，简单的能谱仪是足够cient来表示的荧光共振能量转移效率12。 <ol><li>使用ImageJ软件打开实验文件，选择“插件&gt;微管理”&gt;“打开”微管理文件。 </li><li>运行“FREToffline的”插电式，其中拆分成单个的CFP和YFP通道的时间推移的堆栈。 </li><li>如果背景校正是必需的，这可以使用ImageJ软件进行。 </li><li>点击的YFP堆栈的图像，并选择一个或几个区域使用的“写意选择”工具的兴趣，并把它们添加到“MultiMeasure”插件窗口中按“添加”按钮。 </li><li>选择该地区的利益在“MultiMeasure”的窗口，然后按“多”，获得一个表的每一帧，每个区域的平均灰度值。将数据复制到剪贴板中选择使用Ctrl + A，按Ctrl + C通过按Ctrl + V，打开Excel或地电子表格，并粘贴数据 由程序执行的测量可以被配置在分析&gt;集M<html>的easurements，在这里你可以定义要测量的参数。我们通常只选择“平均灰度值”在此对话框中。 </li><li>点击进入CFP堆栈的图像。执行5.5中所描述的相同。 CFP强度数据粘贴到同一个Excel在地电子表格中。 </li><li>使用Excel或Origin软件，计算修正后的FRET比率。当简单的单链单分子生物传感器的成像，我们修正的供体荧光只为bleedthrough的进入受体通道。在这种情况下，校正后的受主/施主比是： 比率=（YFP - B X CFP）/ CFP 其中，B是可以通过与CFP质粒转染细胞，和测量中的，YFP的信道（B = YFP / CFP），供体荧光的百分比确定的校正因子。省略此单分子生物传感器的校正是可能的，因为它只会影响整体的振幅的FRET的响应没有任何定性的影响的曲线的形状。 </li></ol>jove_title“&gt; 6代表性的成果。 图1示出了一个例子的完全组装FRET成像设置由尼康倒置显微镜，CoolLED，DV2的双屏显示和滨松ORCA-03G CCD照相机。要建立的硬件组件和计算机之间的通信，在I / O Arduino板被连接到计算机，并连接到CoolLED如在图2中所示。要控制光源的相机和图像采集同步方式，微Manager软件必须安装并正确配置（参见图3）。这个免费软件可以很容易地适应各个实验需要添加必要的插件。 图4A显示了具有代表性的原始FRET比率从测量使用的cAMP传感器Epac1营13在293A细胞中表达的跟踪监测β-肾上腺素能激动剂的影响异丙肾上腺素适用于时间点150秒，＆打赌;-受体阻滞剂普萘洛尔在时间点300秒（进行4.3-4.10中描述的）加入。可以分析这些数据脱机并校正到YFP通道所描述的CFP bleedthrough在5.1-5.7，得到校正后的FRET比迹线示于图4B。这代表实验表明在监测的荧光共振能量转移后，异丙肾上腺素治疗的比例，这表明细胞内cAMP增加缓慢下降。普萘洛尔作为β-受体阻滞剂异丙肾上腺素信号反转，导致cAMP的基础水平减少。 FRET信号可以在线监视期间的所有实验（如4.9中所述）中的这些变化。可以进行这样的实验，用各种常用的生物传感器设计为监视不同的第二信使或生化过程。 <img alt="图1" src="/files/ftp_upload/4081/4081fig1.jpg" /> 图1。布局的共振成像设置由一个CoolLED，尼康显微镜，DV2双屏幕显示转换后，ORCA-03G CCD相机。 <img alt="图2" src="/files/ftp_upload/4081/4081fig2.jpg" /> 图2。Arduino的I / O电路板和它的连接。该板被定位在一个自安装的塑料盒。一个标准的BNC电缆连接的LED的引脚8和电路板的GND（0）。 <img alt="图3" src="/files/ftp_upload/4081/4081fig3.jpg" /> 图3。截图展示集成的系统组件使用的硬件配置向导。A）启动硬件配置向导“。B）添加所需的设备中提到的2.3。 <a href="http://www.jove.com/files/ftp_upload/4081/4081fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" src="/files/ftp_upload/4081/4081fig4.jpg" /> 图4。代表FRET实验技术T的措施，cAMP水平在293A细胞转染与Epac1营。首先，刺激细胞的β-肾上腺素能激动剂异丙肾上腺素（100nM的，在第30帧，或在150秒），以增加cAMP（YFP / CFP中减少观察到FRET比）。细胞，随后用β-受体阻滞剂心得安（10μM，60帧或300秒），从而导致一个的FRET比增加，减少环磷酸腺苷A）原在线FRET比率从一个地区的利益相应的痕迹在实验过程中监测到一个单细胞。B）修正后比跟踪进行离线数据分析中所描述5.1-5.7。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gi+protein+activation+in+intact+cells+involves+subunit+rearrangement+rather+than+dissociation.">Gi protein activation in intact cells involves subunit rearrangement rather than dissociation.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 16077-16082 (2003).</li><li>Kotlikoff, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+Ca2++indicators:+using+genetics+and+molecular+design+to+understand+complex+physiology.">Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology.</a> J. Physiol. 578, 55-67 (2007).</li><li>Willoughby, D., Cooper, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+of+cAMP+dynamics.">Live-cell imaging of cAMP dynamics.</a> Nat. Methods. 5, 29-36 (2008).</li><li>Nikolaev, V. O., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+cAMP+synthesis+and+degradation+in+living+cells.">Monitoring of cAMP synthesis and degradation in living cells.</a> Physiology (Bethesda). 21, 86-92 (2006).</li><li>Tanimura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Fluorescence+Resonance+Energy+Transfer-based+Biosensors+for+the+Quantitative+Analysis+of+Inositol+1,4,5-Trisphosphate+Dynamics+in+Calcium+Oscillations.">Use of Fluorescence Resonance Energy Transfer-based Biosensors for the Quantitative Analysis of Inositol 1,4,5-Trisphosphate Dynamics in Calcium Oscillations.</a> J. Biol. Chem. 284, 8910-8917 (2009).</li><li>Nikolaev, V. O., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+techniques+for+real-time+monitoring+of+cGMP+in+living+cells.">Novel techniques for real-time monitoring of cGMP in living cells.</a> Handb. Exp. Pharmacol. , 229-243 (2009).</li><li>Nausch, L. W., Ledoux, J., Bonev, A. D., Nelson, M. T., Dostmann, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+patterning+of+cGMP+in+vascular+smooth+muscle+cells+revealed+by+single+GFP-linked+biosensors.">Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 365-370 (2008).</li><li>Borner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+measurements+of+intracellular+cAMP+concentrations+and+cAMP+analog+permeability+in+intact+cells.">FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells.</a> Nat. Protoc. 6, 427-438 (2011).</li><li>Nikolaev, V. O., Bunemann, M., Hein, L., Hannawacker, A., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+single+chain+cAMP+sensors+for+receptor-induced+signal+propagation.">Novel single chain cAMP sensors for receptor-induced signal propagation.</a> J. Biol. Chem. 279, 37215-37218 (2004).</li><li>Hong, K. P., Spitzer, N. C., Nicol, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+molecular+toolkit+for+cAMP+studies+in+live+cells.">Improved molecular toolkit for cAMP studies in live cells.</a> BMC Res. Notes. 4, 241-24 (2011).</li><li>Niino, Y., Hotta, K., Oka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+live+cell+imaging+using+dual+FRET+sensors+with+a+single+excitation+light.">Simultaneous live cell imaging using dual FRET sensors with a single excitation light.</a> PLoS One. 4, e6036 (2009).</li><li>Palmer, A. E., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+calcium+signaling+using+genetically+targetable+fluorescent+indicators.">Measuring calcium signaling using genetically targetable fluorescent indicators.</a> Nat. Protoc. 1, 1057-1065 (2006).</li><li>Brumbaugh, J., Schleifenbaum, A., Stier, G., Sattler, M., Schultz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-+and+dual-parameter+FRET+kinase+probes+based+on+pleckstrin.">Single- and dual-parameter FRET kinase probes based on pleckstrin.</a> Nat. Protoc. 1, 1044-1055 (2006).</li><li>Aoki, K., Matsuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+small+GTPase+activity+with+fluorescence+resonance+energy+transfer-based+biosensors.">Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors.</a> Nat. Protoc. 4, 1623-1631 (2009).</li></ol>

没有利益冲突的声明。

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 ...

fret-microscopy-for-real-time-monitoring-signaling-events-live-cells

Research

JoVE Journal

Biology

23.0K 次观看.  Georg August University Medical Center, Göttingen, Germany. 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。

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

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

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

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

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

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

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

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

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

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

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

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

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

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

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of 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

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

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

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

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

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

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

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

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 ...