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>

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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...

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 border="0" cellpadding="0" cellspacing="0" width="1083">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Name of Material/ Equipment</td>
			<td style="width:211px;">Company</td>
			<td style="width:211px;">Catalog Number</td>
			<td style="width:443px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Double-sided tape</td>
			<td style="width:211px;">3M</td>
			<td style="width:211px;">--</td>
			<td>~ 75 um thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">EMCCD camera</td>
			<td style="width:211px;">Andor Technology</td>
			<td style="width:211px;">DU-897E-CS0-#BV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Ultrasonic cleaner</td>
			<td style="width:211px;">Branson</td>
			<td style="width:211px;">2510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Fluorescence filter set</td>
			<td style="width:211px;">Chroma</td>
			<td style="width:211px;">49016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Actin polymerization buffer&#160;</td>
			<td style="width:211px;">Cytoskeleton&#160;</td>
			<td style="width:211px;">BSA02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Biotin G-actin</td>
			<td style="width:211px;">Cytoskeleton&#160;</td>
			<td style="width:211px;">AB07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">G-actin&#160;</td>
			<td style="width:211px;">Cytoskeleton&#160;</td>
			<td style="width:211px;">AKL95</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">General actin buffer&#160;</td>
			<td style="width:211px;">Cytoskeleton&#160;</td>
			<td style="width:211px;">BSA01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Laser shutter (with driver)</td>
			<td style="width:211px;">Electro-Optical Products Corp.&#160;</td>
			<td style="width:211px;">SH-10-MP</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">IDL</td>
			<td style="width:211px;">Exelis Visual Information Solutions</td>
			<td style="width:211px;">--</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Neutravidin&#160;</td>
			<td style="width:211px;">Fisher Scientific</td>
			<td style="width:211px;">PI-31000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Coverslip</td>
			<td style="width:211px;">Fisherbrand&#160;</td>
			<td style="width:211px;">22X30-1.5</td>
			<td>0.16-0.19 mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Microscope slide</td>
			<td style="width:211px;">Gold Seal Microslides&#160;</td>
			<td style="width:211px;">30103X1</td>
			<td>0.93-1.05 mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plasma cleaner</td>
			<td style="width:211px;">Harrick Plasma</td>
			<td style="width:211px;">PDC-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Glass bottom dish&#160;</td>
			<td style="width:211px;">In Vitro Scientific</td>
			<td style="width:211px;">D35-20-1.5-N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Cy3-DNA oligos</td>
			<td style="width:211px;">Integrated DNA Technologies</td>
			<td style="width:211px;">--</td>
			<td>5&#39;-Cy3/GCCTCGCTGCCGTCGCCA-3&#39;Bio</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Fluorescent beads&#160;</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">T-7280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Qdot 605-streptavidin&#160;</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">Q10101MP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Qdot605&#160;</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">Q21301MP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Qdot705</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">Q22021MP&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Qdot705 Antibody Conjugation Kit</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:211px;">Q22061MP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Matlab</td>
			<td style="width:211px;">MathWorks</td>
			<td style="width:211px;">--</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Optical table</td>
			<td style="width:211px;">Newport Corp</td>
			<td style="width:211px;">--</td>
			<td>RS4000 Series</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">60X Objective</td>
			<td style="width:211px;">Nikon</td>
			<td style="width:211px;">Plan Apo VC 60x WI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">100X Objective</td>
			<td style="width:211px;">Olympus</td>
			<td style="width:211px;">PlanApo 100X/1.45 Oil &#8734;/0.17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">60X Objective</td>
			<td style="width:211px;">Olympus</td>
			<td style="width:211px;">UPlanApo 60X/1.20W</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Inverted microscope</td>
			<td style="width:211px;">Olympus</td>
			<td style="width:211px;">IX71/IX70/IX81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Origin</td>
			<td style="width:211px;">OriginLab</td>
			<td style="width:211px;">--</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Anti-FLAG antibody</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">F7425-.2MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">ATP</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">A7699</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">BME&#160;</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">63689-25ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">BSA</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">BSA-biotin</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">A8549-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">CK</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">C3755</td>
			<td>Creatine Phosphokinase from rabbit muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">CP</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">P1937</td>
			<td>Phosphocreatine di(tris) salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">DTT</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">43815</td>
			<td>DL-Dithiothreitol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">EGTA</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">E3889</td>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">HCl&#160;</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">93363-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">HEPES&#160;</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">KCl</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">P9333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">MgCl2</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">M1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">NaCl</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">PCA</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">03930590</td>
			<td>Protocatechuic acid&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">PCD</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">P8279</td>
			<td>Protocatechuate-3,4-dioxygenase&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">TMOS</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">341436-25G</td>
			<td>Tetramethyl orthosilicate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Tris-HCl&#160;</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">93363</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Trolox</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:211px;">238813</td>
			<td>6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">1&#8221; diameter broadband dielectric mirrors with mounts</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">BB1-E02, KM100</td>
			<td>Quantity: 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">&#189;&#8221; diameter posts</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">TR4</td>
			<td>Quantity &#8805; 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">10x beam expander</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">BE10M-A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">2&#8221; diameter broadband dielectric mirrors with mounts</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">BB2-E02, KM200</td>
			<td>Quantity: 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">2&#8221; diameter f = 300 mm lens with mount</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">LA1256-A, LMR2</td>
			<td>TIR lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Fluorescent alignment target&#160;</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">VRC2SM1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Laser safety goggles</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">LG3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">ND filter(s)</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">FW1AND</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Optical beam profiler</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">BP209-VIS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Post-mounted iris diaphragm</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">ID25</td>
			<td>Quantity: 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Shearing interferometer</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">SI100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">XYZ translation stage, &#189;&#8221; travel&#160;</td>
			<td style="width:211px;">Thorlabs&#160;</td>
			<td style="width:211px;">T12XYZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Laser</td>
			<td style="width:211px;">World Star Technologies&#160;</td>
			<td style="width:211px;">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?...

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

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

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

科研

生物学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

荧光显微镜下细胞电融合的可视化

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis和显微操作的微循环活体荧光成像

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

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 ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

小鼠小肠的混合原代培养拟肠激素分泌的研究和活细胞成像的肠内分泌细胞的供

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

黄荧光蛋白标记 p62 在攻击样诱导结构中光漂白后的荧光恢复

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...