Name: fluorescence imaging with one-nanometer accuracy (fiona)
Uploaded: 2014-09-26T15:00:00.000+00:00
Duration: 11 min 56 s
Description: Watch this Scientific Journal Video about Fluorescence Imaging with One-nanometer Accuracy FIONA at JoVE.com

这项工作是由美国国立卫生研究院资助068625，美国国家科学基金会资助1063188活细胞0822613.特别感谢滨海诺维奇博士贝克曼研究所高级科学技术对兔眼礼物的物理中心的支持。

道德声明:兔角膜组织收集，根据伊利诺伊州机构动物护理和使用指南的大学。 1 TIRFM设置注:戴激光护目镜所有的时间。 <ol><li>确保在材料清单中列出的所有必要的光学组件可用并准备调整。如果需要，可使用替代品与其他公司同等的功能。确保反射镜和透镜应该有防反射（AR）涂层配套的激光在使用。 </li><li>设置所有的光学元件的高度，以在显微镜背面端口的中心的高度。 </li><li>挂载激光，激光快门和ND滤镜（S）。使用ND过滤器，以衰减激光功率下尽可能低，同时保持光束可见。拧紧相应的六角扳手螺丝。 </li><li>规划的光路，并用胶带或标记的光学平台上（点标记在图1中泰德蓝线）。为简单起见，将沿着光学平台上的孔的线的直的路径。 </li><li>地点镜M1（ 图1）在第一直角转弯。放置在两个膜片沿计划光束路径的第二直线部。同时调整位置和M1的倾斜，使得激光穿过膜片。 </li><li>地方镜M2（ 图1）在第二直角转弯。放置10X光束扩张（L1及L2， 如图1所示 ）沿该路径的第三直部。调整其倾斜使得所述光束扩展器是平行于两个光学表和计划的光束路径。 <ol><li>调整M1和M2的迭代，使得激光进入直行通过扩束器的两个透镜的中心。重复此步骤，直至扩束的束分布曲线是非限幅高斯。 </li></ol></li><li>调节货币供应量M1为中心在L1梁（ 网络gure 1）和M2以迭代地集中在L2上的光束（ 图1）。一个坏的光束分布，通常是指激光裁剪;用一张白纸光束扩展后，以阻断光束以与眼睛检查的束分布曲线。对于高精密的分析，使用光束分析器。 </li><li>调整，使得所述光束被准直的L1和L2之间的距离。重复步骤1.8和1.9，如果有必要的。 注意:当光束尺寸不随距离变化时，梁是一个TIRFM设置足够的准直。为了进一步改善光束准直，如剪切干涉工具也可以使用。扩建后的典型光束尺寸为约20毫米。 </li><li>快门的激光。拧开显微镜物镜和螺杆的荧光对准目标。地方镜M3和M4（ 图1），以指示扩展光束进入显微镜端口和上转台内的分色镜。确保激光束反弹吨他二向色和朝向天花板。 </li><li>调整M3居中在荧光靶的光束的最亮部分和M4来调整波束倾斜进行垂直。 </li><li>快门的激光和螺杆的目标回。如果在前面的步骤中的对准做得好应该有一个对称的点离开的目的。微调M3和M4的倾斜来优化激光功率和光束轮廓出来的目标。 </li><li>挂载一个EMCCD相机的显微镜和相机连接到电脑。启动软件的摄像头。 </li><li>安装在显微镜荧光样品（荧光的溶液）。看相机上的明亮的荧光斑点。检查点不会在屏幕上移动的焦点发生改变。 </li><li>放置在TIR透镜（L3， 图1）上的XYZ平移台在从物镜的后焦面的距离，该距离等于焦距L3的（约30厘米）。调整L3的位置使得激光穿过透镜的中心。 </li><li>翻译L3沿着光束路径来调整光束准直。确保该光束仍集中在显示器上，并左右对称的形状。 注:照明区域在样品平面的增加而递减TIR透镜的焦距。一般情况下，使用最小的焦距，可以容纳上设置。 </li><li>翻译L3垂直于光束路径倾斜的梁的目标。保持翻译的TIR透镜，使得TIR实现。通过EMCCD相机，和微调的L3观察荧光珠样本，以获得良好的信噪比（SNR）。 </li></ol><img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51774/51774fig1highres.jpg" width="500" /> 图的全内反射荧光显微镜（TIRFM）1光学配置。 2，FIONA上的Cy3-DNA <ol><li> ç精益显微镜载玻片和盖玻片:冲洗显微镜载玻片和盖玻片与DDH 2 O和异丙醇和氮气抹干;放置在等离子体清洁的载玻片和盖玻片下氩等离子体5分钟。 </li><li>构造样品室（如描绘于图2）。 <ol><li>将组织在板凳上，然后把一张镜头纸在组织之上。广场上的镜头纸的幻灯片。确保滑干净的一侧朝上。 </li><li>应用两个条双面胶带上沿着长边滑动，更让3-5毫米的中央间隙。放置在载玻片上方的清洗盖玻片。确保盖玻片的清洁面朝幻灯片。 </li><li>用枪头向下压在双面胶带。用剃刀从幻灯片中删除多余的带子，这样带子仍然只在盖玻片。 注:该腔室的开口端保持开路，并作为入口和出让。该腔室体积是几微升。 </li></ol></li></ol><img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51774/51774fig2highres.jpg" width="500" /> 图2:素描典型样品室（一）顶视图。 （二）从右侧视图; （c）从所述前部的侧视图。 <ol start="3"><li>固定在样品室的内表面的Cy3-DNA。 <ol><li>制备T-50的缓冲液（10mM的Tris-HCl pH 8.0的50 mM氯化钠）。制备的BSA-生物素在T-50以1毫克/毫升的最终浓度。通过在10毫克/毫升的最终浓度在T-50溶解BSA制备T50-BSA缓冲液。 </li><li>制备0.5毫克/毫升的中性在T50-BSA缓冲液。制备生物素化的Cy3标记的DNA（Cy3标记的DNA），在T50-BSA在5-10分的最终浓度。 </li><li>移液管将10μl的BSA-生物素（1毫克/毫升）到样品室中。等待5分钟。 </li><li&gt; 40微升T50-BSA洗涤室。移液管将10μl中性（0.5毫克/毫升）到样品室中。孵育5分钟。 </li><li>洗用40微升T50-BSA的腔室。吸管20微升的Cy3-DNA导入样品室。孵育5分钟，然后用80μl的T50-BSA洗涤室。 </li></ol></li><li>图像的Cy3-DNA TIRFM下单分子。 <ol><li>制备成像缓冲液（100微升）混合1μl的儿茶酸，3,4 - 双加氧酶（PCD，5微米），4微升原儿茶酸（PCA，62.5毫摩尔），50微升6羟基2,5,7,8- tetramethylchromane-2-羧酸（Trolox的，2毫米的T-50），和45微升T50-BSA。 </li><li>移液管中加入30μl成像缓冲器，等待8-10分钟。 </li><li>装载样品进行成像上都配有绿色激光（532 nm）的TIRFM，100倍，油浸物镜（1.45 NA）和EMCCD相机。 </li><li>设置曝光时间为100至500毫秒和EM增益，以25〜100收购电影样品的1,000帧。 </li></ol></li><li> FIONA进行数据分析上的Cy3-DNA录制的图像。 <ol><li>确定有效的像素的大小（ 即 ，从像素到纳米转换系数）除以物理像素尺寸的总放大率（在显微镜物镜乘以任何额外的放大倍数的放大倍数）（从EMCCD相机的规格表中读出）。 </li><li>从像素强度确定的转换系数除以CCD的灵敏度（ 即 ，每个A / D计数，来自CCD相机的规格表中读出电子）的电子倍增器（EM）的图像采集过程中使用的增益，以光子数。 </li><li>编译并运行FIONA.pro的FIONA分析。使用该IDL程序导入所获取的图像，以输入的有效像素尺寸（来自步骤2.5.1），并从强度的光子数（来自步骤2.5.2）的转换系数，并且选择的斑点为FIONA分析。 注:在结束时，编程的，米将输出与2D高斯函数，以及总光子数和定位精度的拟合结果。典型的结果示于代表性的结果和图4c-4d的部分。 </li><li>编译并运行phcount.pro表征光子计数。使用该IDL程序来测量光漂白之前由荧光团发射的光子的平均数，从强度到光子数（来自步骤2.5.2）导入所获取的图像和输入的转换因子。 注:然后程序会自动检测荧光斑点（手动选择一个选项），计算出光子数为帧数的函数，输出光子计数的痕迹。 <ol><li>摒弃坏的痕迹和漂白基线校正后指定的帧范围。最后，该方案将输出不被丢弃的总光子数的所有点的列表。然后绘出光子数分布和适合的类分布灰与指数衰减，以获得平均光子数。典型的结果示于的代表性结果的部分和图4E-4023。 </li></ol></li></ol></li></ol> 3，FIONA应用量化电机（ 如肌球蛋白对肌动蛋白）动力学纳米尺度<ol><li>肌动蛋白聚合（ 即准备的F-肌动蛋白）1天FIONA实验前。 <ol><li>重组G-肌动蛋白（单体）和生物素的G-肌动蛋白（单体）到10毫克/毫升与一般的肌动蛋白缓冲液中。搅拌均匀，以确保两者都充分溶解，并在冰上保持两个。 </li><li>将10微升的G-肌动蛋白（单体）与1.7μL生物素的G-肌动蛋白的1.5 ml离心管。加入100μl冰冷的肌动蛋白聚合缓冲区。 </li><li>离开混合一夜之间在4°C（F-肌动蛋白组成），然后添加DDH 2 O为1毫升总体积。 </li><li>存储该肌动蛋白丝（F-肌动蛋白）在4℃下进行实验的以后使用。 注:花丝会瓦解，并缩短了时间，但可用于在至少两个星期。 </li></ol></li><li>制备样品的成像。 <ol><li>做一个样品室（如在协议2.1和2.2中所述）。移液管在20μl在DDH 2 O的生物素化的BSA在1毫克/毫升孵育10分钟。用清水冲洗30微升DDH 2 O。 注:此块玻璃表面，并规定了对生物素的肌动蛋白丝结合。生物素化的聚-L-赖氨酸 - 聚乙二醇（PLL-PEG），可以提供相同的功能。 </li><li>移液管在0.5毫克/毫升的中性。孵育2分钟，然后用30微升M5缓冲洗室。 </li><li>移液管中制备的F-肌动蛋白稀释25倍于一般的肌动蛋白的缓冲液中，至终浓度为〜0.004毫克/毫升。等待10分钟，用清水冲洗30微升缓冲室。 </li><li>肌球蛋白稀释弗吉尼亚州与FLAG标签M5的缓冲液（20毫米HEPES（pH值7.6），2毫米氯化镁2，25 mM的氯化钾，1毫米EGTA）30倍至终浓度Ö˚F为250牛米。混合1μl的肌球蛋白与1μl的抗-FLAG-Qdot705（〜1微米，从抗​​FLAG抗体和Qdot705使用Qdot705抗体偶联试剂盒根据来自制造商的使用说明书偶联）。添加8微升M5，以填补至10微升。移液器上下拌匀。孵育在冰上10分钟。 注意:这将产生1电机4量子点混合，在约25纳米肌球蛋白的浓度。 </li></ol></li><li>成像肌球蛋白走在肌动蛋白。 <ol><li>通过混合制备成像缓冲液（100微升），84微升M5-BSA（M5缓冲用1毫克/毫升BSA），1μl的ATP（50μM的在DDH 2 O），2微升的DTT（500mM的在DDH 2 O），1微升CK（500单位/毫升），5微升的CP（200毫摩尔），1微升的PCD，4μl的PCA，1μl的肌球蛋白的Qdot后稀释另一个10倍至2.5纳米的肌球蛋白的浓度，和1微升BME。 </li><li>移液管在20μl成像缓冲器到样品室，并孵育​​8-10分钟。 </li><li>图片对T的样本IRF显微镜在30毫秒的曝光。获得至少1000架。如有必要，调整肌球蛋白量子点的体积在步骤3.3.1。 </li></ol></li><li>执行数据分析并发现的肌球蛋白的步行的步长。 <ol><li>在ImageJ的17打开视频文件，裁剪左右移动现场视频。裁剪一个足够大的区域，该点永远不会在20个像素边缘，并确保有视频没有其它景点。确保这个地方正在沿直线路径。 </li><li>通过视频跟踪点来生成x和y坐标通过时间，以像素为单位，通过施加FIONA分析（步骤2.5.3）每个和视频的每一帧。 </li><li>转换像素以纳米，如前一节中所描述。 </li><li>从初始位置作为时间的函数计算位移。 </li><li>上运行的位移t检验，得到的肌球蛋白行走的步骤。 注:为t检验（step_t_test.zip）程序编码在IDL和CON14子程序的文件夹中sists。 <ol><li>打开所有在IDL中的子程序和编译所有的两倍。然后运行mtltyanalysis_ttest.pro并选择只包含在单个列的距离数据的文本文件。一个输出的Excel文件，将会产生一个包含原始数据的拟合，并且步长大小。 </li></ol></li><li>从步长列删除所有零值。剧情采用原产地或MATLAB的步长的分布。符合高斯直方图。 注:零值的步长​​大小需要被删除，因为零的步骤意味着没有步骤是从先前帧。嵌合给出大约36纳米的峰值（ 如图5）。 </li></ol></li></ol> 4，厚样品制备FIONA <ol><li>制备包封在溶胶 - 凝胶量子点。 <ol><li>混合4.5毫升TMOS 1毫升DDH 2 O和100微升的HCl（120毫米）。在超声波清洁器SP超声处理在冰上将混合物搅拌30分钟ecified在材料清单（频率为4​​0千赫，加热= OFF）。混合溶液中，每10分钟。 </li><li>稀释1.5μLQdot605在1.5ml HEPES（50 mM的pH值为7.2）。混合溶液用1.5ml TMOS来自之前的步骤。 </li><li>把混合物倒入玻璃底菜。密封的玻璃底菜用封口膜，并储存在4°C下1.5小时。 </li><li>在PBS中，加入2毫升1.0％BME到样品盘中，在室温下孵育成像之前30分钟。 </li></ol></li><li>准备沾上量子点角膜样本。 注:兔子的眼睛是从滨海诺维奇博士的礼物。 <ol><li>从眼睛中分离角膜切成3mm×3mm的片。 </li><li>稀释1微升量子点605-链霉亲和1毫升的PBS。培养角膜组织与1nM量子点在4溶液 ºC1小时。洗，用PBS将组织。 </li><li>取一干净的载玻片和＃1.5盖玻片。把4层的双面胶带上的载玻片上沿长边，次把另一个4层的双面胶带平行于前面的磁带，并留下一个通道大约1厘米之间。将组织从在通道的中间的前面的步骤，并用盖玻片覆盖。 </li><li>用手指轻压盖玻片的两侧，以使其粘在胶带。前成像湿用50微升的PBS频道。 </li></ol></li><li>图像量子点中的溶胶 - 凝胶和角膜。 <ol><li>对于FIONA成像厚的样品中，使用60X水浸泡的目的与工作0.27毫米，或与工作为0.28mm的距离的60倍水浸泡目标的距离。 </li><li>安装在显微镜的样品。调整TIRF透镜，使得其到达落射荧光模式（ 即激光束出来的目标与对盖玻片的角度）。插入一个额外的放大倍率镜头（3.3X或4.0X）。 </li><li>移动物镜的焦平面至所需的z轴位置（ 如 &gt; 200微米）。动的影像记录量子点样品中。 </li></ol></li><li>如本协议第2.5节进行FIONA分析。 </li></ol>

<ol>
	<li>Abbe, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Relation+of+Aperture+and+Power+in+the+Microscope.">The Relation of Aperture and Power in the Microscope.</a> Journal of the Royal Microscopical Society. 2 (3), 300-309 (1882).</li><li>Abbe, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Relation+of+Aperture+and+Power+in+the+Microscope+(continued).">The Relation of Aperture and Power in the Microscope (continued).</a> Journal of the Royal Microscopical Society. 2 (4), 460-473 (1882).</li><li>Thompson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+Nanometer+Localization+Analysis+for+Individual+Fluorescent+Probes.">Precise Nanometer Localization Analysis for Individual Fluorescent Probes.</a> Biophysical Journal. 82 (5), 2775-2783 (2002).</li><li>Yildiz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+V+Walks+Hand-Over-Hand:+Single+Fluorophore+Imaging+with+1.5-nm+Localization.">Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization.</a> Science. 300 (5628), 2061-2065 (2003).</li><li>Yildiz, A., Tomishige, M., Vale, R. D., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+Walks+Hand-Over-Hand.">Kinesin Walks Hand-Over-Hand.</a> Science. 303 (5658), 676-678 (2004).</li><li>Yildiz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+VI+Steps+via+a+Hand-over-Hand+Mechanism+with+Its+Lever+Arm+Undergoing+Fluctuations+when+Attached+to+Actin.">Myosin VI Steps via a Hand-over-Hand Mechanism with Its Lever Arm Undergoing Fluctuations when Attached to Actin.</a> Journal of Biological Chemistry. 279 (36), 37223-37226 (2004).</li><li>Yildiz, A., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Imaging+with+One+Nanometer+Accuracy:+Application+to+Molecular+Motors.">Fluorescence Imaging with One Nanometer Accuracy: Application to Molecular Motors.</a> Accounts of Chemical Research. 38 (7), 574-582 (2005).</li><li>Toprak, E., Yildiz, A., Hoffman, M. T., Rosenfeld, S. S., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+kinesin+is+so+processive.">Why kinesin is so processive.</a> Proceedings of the National Academy of Sciences. 106 (31), (2009).</li><li>Kural, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+melanosomes+inside+a+cell+to+study+molecular+motors+and+their+interaction.">Tracking melanosomes inside a cell to study molecular motors and their interaction.</a> Proceedings of the National Academy of Sciences. 104 (13), 5378-5382 (2007).</li><li>Gordon, M. P., Ha, T., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+high-resolution+imaging+with+photobleaching.">Single-molecule high-resolution imaging with photobleaching.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (17), 6462-6465 (2004).</li><li>Qu, X., Wu, D., Mets, L., Scherer, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometer-localized+multiple+single-molecule+fluorescence+microscopy.">Nanometer-localized multiple single-molecule fluorescence microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (31), 11298-11303 (2004).</li><li>Churchman, L. S., &#214;kten, Z., Rock, R. S., Dawson, J. F., Spudich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+molecule+high-resolution+colocalization+of+Cy3+and+Cy5+attached+to+macromolecules+measures+intramolecular+distances+through+time.">Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (5), 1419-1423 (2005).</li><li>Huang, B., Wang, W., 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=Three-Dimensional+Super-Resolution+Imaging+by+Stochastic+Optical+Reconstruction+Microscopy.">Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy.</a> Science. 319 (5864), 810-813 (2008).</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 (10), 793-796 (2006).</li><li>Bates, M., Huang, B., Dempsey, G. T., 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=Multicolor+Super-Resolution+Imaging+with+Photo-Switchable+Fluorescent+Probes.">Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes.</a> Science. 317 (5845), 1749-1753 (2007).</li><li>Betzig, E., et al. <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 (5793), 1642-1645 (2006).</li><li>Abramoff, M. D., Magalh&#227;es, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+with+ImageJ.">Image processing with ImageJ.</a> Biophotonics international. 11 (7), 36-42 (2004).</li><li>Enderlein, J., Toprak, E., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+effect+on+position+accuracy+of+fluorophore+localization.">Polarization effect on position accuracy of fluorophore localization.</a> Optics Express. 14 (18), 8111-8120 (2006).</li><li>Cheezum, M. K., Walker, W. F., Guilford, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+comparison+of+algorithms+for+tracking+single+fluorescent+particles.">Quantitative comparison of algorithms for tracking single fluorescent particles.</a> Biophysical Journal. 81 (4), 2378-2388 (2001).</li><li>Rasnik, I., McKinney, S. A., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonblinking+and+long-lasting+single-molecule+fluorescence+imaging.">Nonblinking and long-lasting single-molecule fluorescence imaging.</a> Nature Methods. 3 (11), 891-893 (2006).</li><li>Zhuang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Single-Molecule+Study+of+RNA+Catalysis+and+Folding.">A Single-Molecule Study of RNA Catalysis and Folding.</a> Science. 288 (5473), 2048-2051 (2000).</li><li>Aitken, C. E., Marshall, R. A., Puglisi, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Oxygen+Scavenging+System+for+Improvement+of+Dye+Stability+in+Single-Molecule+Fluorescence+Experiments.">An Oxygen Scavenging System for Improvement of Dye Stability in Single-Molecule Fluorescence Experiments.</a> Biophysical Journal. 94 (5), 1826-1835 (2008).</li></ol>

作者宣称，他们没有竞争的经济利益。

FIONA是一种技术来定位具有纳米精度和时间分辨率的荧光发射（有机荧光团或量子点）的位置下降到1毫秒4-8。当足够的光子被收集，该技术允许对多比衍射极限（约200纳米）更精确地确定的荧光发射器的位置，因此该方法将打开的方式来观察哪些还没有被看到与常规的/传统的光学显微镜4 - 8。自从发明，FIONA已成功应用于观察走路多分子马达，如肌球蛋白和驱动蛋白。最近，连同其他工作3,19，促成了诸如暴风影音和Palm 13-16某些新兴的超分辨率技术的国产化进程。 实现FIONA的关键步骤在于收集光子的数目，这是受各种˚F演员。例如，用于FIONA实验的TIRFM设置应该很好对准，使得一个良好的PSF（ 即未拉伸的，未烙印， 等等 ）被实现的，并得到一个合理的信号-噪声比。此外，具有高NA值的物镜应该以收集尽可能多光子尽可能使用。 收集更多的光子，从而增加定位精度，不同的氧清除剂的方法和试剂已探索抑制漂白和闪烁20-22。两种不同的氧清除剂的方法已被用在我们的实验室中。一个是"gloxy"溶液（葡萄糖氧化酶和过氧化氢 ​​酶）20。另一种是PCA / PCD上述22。后的前作品立即混合，但该溶液的pH值随时间的变化。后者保持溶液化学上保持不变，但需要8到10分钟的温育时间。 ás这里示出，但也可以延长FIONA用于使用60X水浸物镜与NA = 1.2厚的样品。这个目标比以前使用的100倍油浸物镜（0.17 MM）长工作距离（0.27毫米）。工作在厚的样品，落射荧光显微术需要被使用。虽然TIRFM的低背景的优势被牺牲，纳米定位精度仍然是可以实现的，而用明亮的量子点。这个扩展将是厚组织成像和医疗应用。 FIONA的应用在几个方面受到限制。首先，作为该与定位精度取决于所收集的光子的总数，FIONA的时间分辨率通常受到损害。其次，FIONA独自只能适用于稀疏标记的样品。换句话说，FIONA会失败，如果多个荧光团是足够接近的，例如它们的PSF重叠。此外，发射的光的散射限制了APFIONA的厚组织成像折叠术。

围绕1882，阿贝发现，可见光显微镜的分辨率为〜λ/ 2NA，或〜200纳米（其中，λ是波长，NA是数值孔径）1,2。因此，比该尺寸更小的任何对象将显示为在光学显微镜衍射极限的光点。然而，这是可能的，以确定中心的点，即，该对象的位置，以更高的精度3。荧光成像与纳米精度（FIONA）是一个简单而有用的技术，本地化单个荧光基团与纳米精度在XY平面4。定位的精度，σμ（ 即 ，平均值的标准误差），取决于所收集的光子的总数， <img alt="式（1）" fo:content-width="1in" src="/files/ftp_upload/51774/intro_eqn.jpg" width="100" /> ，其中N是光子计数，s是荧光点的标准偏差，a是成像检测器的像素尺寸，而b是背景3,4的标准偏差。对于荧光发光〜10,000的光子，FIONA可以实现〜1nm的精度4。 FIONA可以用来精确地确定一个固定的发射器的位置或移动一个（假设图像可采取足够快）。 FIONA可以顺序地被施加到影片的帧，从而跟踪单分子4-8的动作。光保护性试剂可以是必要的，以确保样品不光降解。此外，该荧光物本身可以是任何尺寸的，比衍 ​​射LIMIT- 例如更小或更大，它可以由细胞器（〜1微米）与分散在其膜的许多荧光蛋白。使用FIONA仍然可以得到非常精确的（纳米）平均平均中心的质量的。在定位精度的巨大进步菲奥娜可以解决nanome三元规模的变动随着时间的推移。这已使显微镜到分子尺度4-8。 因为它的发明中，已开发FIONA的变体。例如，明场成像与纳米精度（bFIONA）9，FIONA轻微变形，图像和本地化致密的对象，如黑色素在体内 （深含黑色素的对象）与透射光。此外，FIONA已被用来解决多种染料。例如，单分子的高分辨率成像光漂白（虾）10,11或单分子高分辨率的共定位（SHREC）12已发展到在约10nm的解决两种染料。 （注意，这是分辨率，即可以如何准确地告诉完全相同染料分开。）最近，FIONA分析，以一定的超分辨率显微镜国产化进程作出了贡献，如随机光学RECOnstruction显微镜（STORM）13 - 15和光激活定位显微镜（PALM）16，其中临时暗的荧光团被激发，然后在荧光被本地化。由染料的反复刺激的相当低的浓度（小于每衍射受限斑），然后收集的荧光，通过FIONA他们每个人的分析，我们可以建立一个高分辨率的地图。分辨率则只是受限于光子的数量每种染料推出，以及类似的东西在采集过程中保持静止的样品（包括， 例如 ，在显微镜载物台）。 在本文中，FIONA技术的总结，并简要介绍了已使用FIONA报道进行研究的例子。首先，如何设置所需的设备FIONA实验， 也就是说 ，全内反射荧光显微镜（TIRFM），与上对准光学元件的细节进行说明。那么如何进行简单的FIONA实验上定位固定的Cy3-DNA单分子使用合适的协议，说明。在此之后，利用FIONA的测量单个截头肌球蛋白弗吉尼亚马达标有量子点的36纳米步长给出。肌球蛋白Va为一个重要的processive马达蛋白携带蜂窝货物而沿微丝易位。这里球蛋白弗吉尼亚州建造截断用来删除域无关的步长，并与FLAG标签添加到C端，使其很容易与功能化与抗FLAG抗体的量子点标记的。该实验是在低的ATP进行减缓肌球蛋白和允许使用足够长的曝光时间以获得良好的光子计数在每帧。任何足够亮的荧光标记物可被取代的以下协议。最后，报道近期扩大菲奥娜厚样本应用程序的努力。作为一种证明性的原则，量子点都湿透了在溶胶 - 凝胶和兔眼的角膜，然后成像并使用FIONA本地化。对于成像，具有呐60X水浸物镜= 1.2被使用，因为这一目标具有较长的工作距离比以前使用100倍油浸物镜。以补偿在放大的损失中的目标，一个额外的放大倍率透镜（3.3X或4.0X）插入在发射路径。此外，落射荧光（未TIR）镜需要被用来访问深区域中的厚样品中。它示出在溶胶 - 凝胶和在兔眼的角膜（Z&gt; 200微米），可以用2-3 nm的精度被定位浸泡深量子点。

<table><tbody><tr><td>Double-sided tape</td><td>3M</td><td></td><td>~75 &amp;#181;m thick</td></tr><tr><td>EMCCD camera</td><td>Andor Technology</td><td>DU-897E-CS0-#BV</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>Branson</td><td>2510</td><td></td></tr><tr><td>Fluorescence filter set</td><td>Chroma</td><td>49016</td><td></td></tr><tr><td>Actin polymerization buffer&amp;nbsp;</td><td>Cytoskeleton&amp;nbsp;</td><td>BSA02</td><td></td></tr><tr><td>Biotin G-actin</td><td>Cytoskeleton&amp;nbsp;</td><td>AB07</td><td></td></tr><tr><td>G-actin&amp;nbsp;</td><td>Cytoskeleton&amp;nbsp;</td><td>AKL95</td><td></td></tr><tr><td>General actin buffer&amp;nbsp;</td><td>Cytoskeleton&amp;nbsp;</td><td>BSA01</td><td></td></tr><tr><td>Laser shutter (with driver)</td><td>Electro-Optical Products Corp.&amp;nbsp;</td><td>SH-10-MP</td><td></td></tr><tr><td>IDL</td><td>Exelis Visual Information Solutions</td><td></td><td></td></tr><tr><td>Neutravidin&amp;nbsp;</td><td>Fisher Scientific</td><td>PI-31000</td><td></td></tr><tr><td>Coverslip</td><td>Fisherbrand&amp;nbsp;</td><td>22X30-1.5</td><td>0.16-0.19 mm thick</td></tr><tr><td>Microscope slide</td><td>Gold Seal Microslides&amp;nbsp;</td><td>30103X1</td><td>0.93-1.05 mm thick</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-001</td><td></td></tr><tr><td>Glass bottom dish&amp;nbsp;</td><td>In Vitro Scientific</td><td>D35-20-1.5-N</td><td></td></tr><tr><td>Cy3-DNA oligos</td><td>Integrated DNA Technologies</td><td></td><td>5&#039;-Cy3/GCCTCGCTGCCGTCGCCA-3&#039;Bio</td></tr><tr><td>Fluorescent beads&amp;nbsp;</td><td>Invitrogen</td><td>T-7280</td><td></td></tr><tr><td>Qdot 605-streptavidin&amp;nbsp;</td><td>Invitrogen</td><td>Q10101MP</td><td></td></tr><tr><td>Qdot605&amp;nbsp;</td><td>Invitrogen</td><td>Q21301MP</td><td></td></tr><tr><td>Qdot705</td><td>Invitrogen</td><td>Q22021MP&amp;nbsp;</td><td></td></tr><tr><td>Qdot705 Antibody Conjugation Kit</td><td>Invitrogen</td><td>Q22061MP</td><td></td></tr><tr><td>MATLAB</td><td>MathWorks</td><td></td><td></td></tr><tr><td>Optical table</td><td>Newport Corp</td><td></td><td>RS4000 Series</td></tr><tr><td>60X Objective</td><td>Nikon</td><td>Plan Apo VC 60x WI</td><td></td></tr><tr><td>100X Objective</td><td>Olympus</td><td>PlanApo 100X/1.45 Oil &amp;infin;/0.17</td><td></td></tr><tr><td>60X Objective</td><td>Olympus</td><td>UPlanApo 60X/1.20W</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX71/IX70/IX81</td><td></td></tr><tr><td>Origin</td><td>OriginLab</td><td></td><td></td></tr><tr><td>Anti-FLAG antibody</td><td>Sigma Aldrich</td><td>F7425-.2MG</td><td></td></tr><tr><td>ATP</td><td>Sigma Aldrich</td><td>A7699</td><td></td></tr><tr><td>BME&amp;nbsp;</td><td>Sigma Aldrich</td><td>63689-25ML-F</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td></td></tr><tr><td>BSA-biotin</td><td>Sigma Aldrich</td><td>A8549-10MG</td><td></td></tr><tr><td>CK</td><td>Sigma Aldrich</td><td>C3755</td><td>Creatine Phosphokinase from rabbit muscle</td></tr><tr><td>CP</td><td>Sigma Aldrich</td><td>P1937</td><td>Phosphocreatine di(tris) salt</td></tr><tr><td>DTT</td><td>Sigma Aldrich</td><td>43815</td><td>DL-Dithiothreitol</td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E3889</td><td>Ethylene glycol-bis(2-aminoethylether)-&lt;em&gt;N,N,N&amp;prime;,N&amp;prime;&lt;/em&gt;-tetraacetic acid</td></tr><tr><td>HCl&amp;nbsp;</td><td>Sigma Aldrich</td><td>93363-500G</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma Aldrich</td><td>H0887</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9333</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M1028</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653</td><td></td></tr><tr><td>PCA</td><td>Sigma Aldrich</td><td>03930590</td><td>Protocatechuic acid&amp;nbsp;</td></tr><tr><td>PCD</td><td>Sigma Aldrich</td><td>P8279</td><td>Protocatechuate-3,4-dioxygenase&amp;nbsp;</td></tr><tr><td>TMOS</td><td>Sigma Aldrich</td><td>341436-25G</td><td>Tetramethyl orthosilicate</td></tr><tr><td>Tris-HCl&amp;nbsp;</td><td>Sigma Aldrich</td><td>93363</td><td></td></tr><tr><td>Trolox</td><td>Sigma Aldrich</td><td>238813</td><td>6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid</td></tr><tr><td>1&amp;rdquo; diameter broadband dielectric mirrors with mounts</td><td>Thorlabs&amp;nbsp;</td><td>BB1-E02, KM100</td><td>Quantity: 2</td></tr><tr><td>&amp;frac12;&amp;rdquo; diameter posts</td><td>Thorlabs&amp;nbsp;</td><td>TR4</td><td>Quantity &amp;ge; 6</td></tr><tr><td>10X beam expander</td><td>Thorlabs&amp;nbsp;</td><td>BE10M-A</td><td></td></tr><tr><td>2&amp;rdquo; diameter f = 300 mm lens with mount</td><td>Thorlabs&amp;nbsp;</td><td>LA1256-A, LMR2</td><td>TIR lens</td></tr><tr><td>Fluorescent alignment target&amp;nbsp;</td><td>Thorlabs&amp;nbsp;</td><td>VRC2SM1</td><td></td></tr><tr><td>Laser safety goggles</td><td>Thorlabs&amp;nbsp;</td><td>LG3</td><td></td></tr><tr><td>ND filter(s)</td><td>Thorlabs&amp;nbsp;</td><td>FW1AND</td><td></td></tr><tr><td>Optical beam profiler</td><td>Thorlabs&amp;nbsp;</td><td>BP209-VIS</td><td></td></tr><tr><td>Post-mounted iris diaphragm</td><td>Thorlabs&amp;nbsp;</td><td>ID25</td><td>Quantity: 2</td></tr><tr><td>Shearing interferometer</td><td>Thorlabs&amp;nbsp;</td><td>SI100</td><td></td></tr><tr><td>XYZ translation stage, &amp;frac12;&amp;rdquo; travel&amp;nbsp;</td><td>Thorlabs&amp;nbsp;</td><td>T12XYZ</td><td></td></tr><tr><td>Laser</td><td>World Star Technologies&amp;nbsp;</td><td>TECGL-30</td><td>532 nm, 30 mW</td></tr></tbody></table>

fluorescence imaging with one-nanometer accuracy (fiona)

荧光成像与纳米精度（FIONA）是一个简单而有用的技术，本地化单个荧光基团与纳米精度在XY平面上。在这里，FIONA技术的总结报告，并已使用FIONA进行研究的例子进行了简要介绍。首先，如何设置所需的设备FIONA实验， 也就是说 ，全内反射荧光显微镜（TIRFM），与上对准光学元件的细节进行说明。那么如何进行一个简单的FIONA实验的本地化固定的Cy3-DNA单分子使用合适的协议，其次是利用FIONA来衡量一个截断的肌球蛋白弗吉尼亚州电机标有量子点的36纳米的步长，说明。最后，据报道最近努力FIONA的适用范围扩大至厚的样品。它表明，用水浸物镜和量子点浸泡深在溶胶 - 凝胶和兔眼的角膜（&gt;200微米），为2-3纳米的定位精度都可以实现。

一个典型的客观型TIRFM设置示于图3，首先，表面固定的Cy3-DNA样品进行成像。一个典型的图像示于图4a。该图像是用曝光时间0.5秒，用EM增益= 50，CCD灵敏度= 12.13的摄像机。单一的Cy3-DNA分子的点扩散函数（PSF）是如图4b所示 （由图4a中的箭头所指示的点），其中显色条显示的像素强度的比例。实际的光子计数能够通过由转换系数，α= CCD灵敏度/ EM增益相乘的像素强度来计算。这点包含大约14,000个光子（校正背景后）。 涤纶短纤，然后装上二维高斯函数f（X，Y）= Z 0 + A·EXP（ - （X-μX）2 /（2S×2） - （Y - ＆＃181，Y）2 /（2S Y 2））所示， 图4c（在图4d所示的嵌合残差）。定位的精确度，然后通过计算出的<img alt="式（2）" fo:content-width="2in" src="/files/ftp_upload/51774/results_eqn.jpg" width="200" />其中，i = x或y，S i是嵌合的标准偏差，N是总光子数，a是像素尺寸，并且b是背景的标准偏差。在这个具体的例子中，N = 14528，A = 106.67纳米，S X = 115.5毫微米，S Y = 109.4纳米，B = 18.9，导致σ的定位精度X = 1.3 nm和σY = 1.2纳米。荧光团的定位精度大致正比于1 /√N， 即更多的光子，就越精确定位是。然而，在FIONA的实际应用中，实验观察时间是另一个考虑因素。因此，人们一般应实现的定位精度和观测时间，并计划未来之间的权衡。在这种情况下，它通常是有用的，以确定有多少个光子的总荧光团是能够漂白之前发射。的光子数与帧的典型踪迹示于图4e中 。一个指数拟合给出的平均光子数〜1.4×10 6（ 图4F）。 在数据分析过程中的肌球蛋白的步长测量的示于图5中 ，首先，具有良好的信号与噪声的，因为它走的单个肌球蛋白的视频文件沿着肌动蛋白丝被捕获， 图5a示出了三帧从视频在100毫秒曝光100X油浸物镜拍摄。移动PSF然后通过使用写在IDL中提取的距离随时间变化的信息的自定义代码，它通过T检验将裁剪视频文件追踪对于步骤。 图5b所示的距离随时间的变化（红色）和步取景器输出（白色）。在每个帧中的定位误差掩盖了微量的阶梯形状，因此它达到在每一个对应于定位误差小于半个人们希望看到的理论级大小的帧的光子计数是至关重要的。 图5c示出由数个步骤痕迹组合成一个高斯分布在真正的肌球蛋白步长一个直方图。高斯拟合直方图箱产生的最后一个步骤大小35.8±0.4 nm的测量。 因为溶胶 - 凝胶和角膜样品是透明的，激发激光器可深入样品而不被散射太多。此外，自发荧光的样品被最小化。当量子点的低浓度标记，能够收集来自量子点荧光深在高信噪比的样品。该使用水客观的给我们的270或280微米的工作距离，这意味着它能够集中尽可能280微米距离的盖玻片。这使我们能够进行FIONA分析厚的样品在量子点。对于量子点的溶胶-凝胶样品中，附近的盖玻片1-2 nm和280微米深入样品（ 图6a-6b）中 2-3处的定位精度得以实现。用于在生物样品中的量子点（从兔眼的角膜的一部分， 图6E），附近的盖玻片1-2 nm和2-3处的定位精度在〜223微米深入样品（ 图6C-6D）实现。应该注意的是定位精度是通过使用额外的放大倍率透镜，没有这些得到的6-7纳米的定位精度提高。这是与以前的数值研究显示，定位精度可以通过从大约200改变有效像素尺寸可以提高相一致纳米至〜即使收集光子的总数可能是低级由于附加的反射/折射18为50nm。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51774/51774fig3highres.jpg" width="500" /> 图3的光学结构。全内反射荧光（TIRF）显微镜的光学配置。a）是图像时的激光是在TIRF条件，和b）是激光在天花板上的光束形状时的激光是在落射照明。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51774/51774fig4highres.jpg" width="500" /> 图4本地化固定的Cy3的DNA。一）CCD图像（256×256像素）的Cy3-DNA，B）单一的Cy3 DNA分子的CCD图像由YEL表示在一个）。C低箭头）安装单一的Cy3 DNA分子的点扩散函数与二维高斯函数。 四）从C拟合残差）。 五）光子计数与帧数。的F）的分配漂白前通过的Cy3分子发射的光子的数量。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51774/51774fig4highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51774/51774fig5highres.jpg" width="500" /> 菲奥娜图5球蛋白行走观察。 a）由实施例的数据文件的帧对应于t = 0秒，30秒和60秒。肌球蛋白-量子点结构正朝着沿直线路径，并具有良好的光子计数（&gt; 5000）中的每一帧。 二）FIONA应用到每一帧一愕LDS是绘制在红色的距离随时间变化的痕迹。基于该T-检验的工序寻找算法被用来然后提取各步骤，并输出叠加在白色。步长被标记在白色中纳米。 三）从多条迹线的步骤组合成一个直方图单元。测得的步长是高斯分布约35.8±0.4纳米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51774/51774fig5highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51774/51774fig6highres.jpg" width="500" /> 图6上的量子点深厚的样品在FIONA分析。QD 605的溶胶- 凝胶 ）荧光图像在Z = 280微米和90X的放大倍率。 二）QD的点扩散函数标记的）。 σX = 2。6纳米，σY = 2.3纳米。在X轴和Y轴的每个单元表示在兔角膜组织在Z = 223微米和360X放大倍率。 四）QD的PSF标志着三）266.7纳米。 三）荧光量子点655的图像。 σX = 2.2纳米，σY = 3.6纳米。在X和Y轴上的每个单位代表44.4纳米。 五）图片角膜组织安装在盖玻片， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51774/51774fig6highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Fluorescence Imaging with One-nanometer Accuracy FIONA at JoVE.com

Fluorescence Imaging with One-nanometer Accuracy FIONA

单荧光团可与纳米精度利用FIONA本地化。这里的FIONA技术的总结报告，以及如何进行FIONA实验进行说明。

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

fluorescence-imaging-with-one-nanometer-accuracy-fiona

Universidad San Sebastian

Research

JoVE Journal

Biology

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

17.6K 次观看.  University of Illinois at Urbana-Champaign. 单荧光团可与纳米精度利用FIONA本地化。这里的FIONA技术的总结报告，以及如何进行FIONA实验进行说明。 

视频: Fluorescence Imaging with One-nanometer Accuracy FIONA

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

紧凑型量子点的单分子成像

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

科研

工程学

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

发育生物学

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

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

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., ...

生物工程

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

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

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 ...

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

可视化单分子络合物在体内使用高级荧光显微

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

生物学

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

细菌细胞的三维成像，实现精确的细胞表现和精确的蛋白质定位

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

免疫与感染

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

超分辨率成像细菌科机械

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...