我们承认贾斯汀平克尼和约翰内斯Hohlbein的帮助，定制显微镜和谢默斯·霍顿的建设，为本地化的软件。罗德里戈·雷耶斯-拉莫特被感谢提供E。大肠杆菌菌株。该研究是由欧盟委员会第七框架计划资助FP7/2007-2013 HEALTH-F4-2008-201418，英国生物技术与生物科学研究理事会资助BB/H01795X/1，欧洲研究理事会资助261227至ANK。 DJS是由威康信托基金项目资助WT083469。苏是由MathWorks公司博士后奖学金支持。

1。细胞培养使用无菌培养管和移液器吸头。 大肠杆菌大肠杆菌菌株AB1157 POLA-PAmCherry携带POL 1的C-末端PAmCherry融合。融合通过使用λ-Red重组作为Datsenko 等人取代野生型基因插入到本机的染色体位置。融合蛋白的8功能性被证实作为判断细胞的生长速率和灵敏度的DNA损伤剂甲基磺酸甲酯（MMS）。在建筑的细胞株的更多信息可在Uphoff 等人 7可以发现，Datsenko 等人，8，和 雷耶斯-拉莫思等 9细胞培养物生长在M9基本培养基中，以减少自发荧光和避免对显微镜载片上的背景颗粒。可选地，一种营养丰富的确定的培养基中可使用10。 <ol><li>连胜大肠杆菌大肠杆菌菌株AB1157 POLA-PAmCherry从一个卢里亚肉汤（LB）琼脂板选择性抗生素（在这里，25微克/毫升卡那霉素）冷冻甘油和孵化在37℃过夜。 </li><li>从单细胞集落接种5ml的LB培养和生长，在37℃下振荡以220rpm搅拌3小时。 </li><li>稀释培养1:10,000到5ml基本培养基（M9培养基，MEM氨基酸+脯氨酸，MEM维生素，0.2％甘油）中并在37℃下振荡以220rpm过夜。 </li><li>第二天早晨，用分光光度计测定光密度（OD），并稀释的培养在5毫升的新鲜最小培养基至OD 0.025。生长2小时，在37℃以220rpm振荡早期指数生长期（OD值0.1）。 </li><li>集中1毫升细胞在1.5ml微量离心管中离心2300×g离心5分钟。去除上清，重悬细胞沉淀在20微升残留培养基中，振荡。 </li></ol> 2。显微镜幻灯片Preparat离子<ol><li>准备在DH 2 O 1.5％的低荧光琼脂糖溶液使用微波炉融化琼脂糖直至溶液澄清。通过轻轻地上下吹打几次混匀500μl的熔化的琼脂糖溶液与500μl的2×基础培养基中。 </li><li>均匀地涂抹在琼脂糖溶液在显微镜盖玻片（否1.5厚）的中心。这必须尽快进行琼脂糖冷却之前，避免气泡。 </li><li>压平垫与第二盖玻片（否1.5厚度）。以消除背景荧光颗粒，盖玻片预先在炉中燃烧，在500℃下进行1小时。烧盖玻片可保存周在室温下以铝箔覆盖。 </li><li>对于DNA损伤的实验中，制备含100毫米彩信琼脂糖垫。按照程序步骤2.1-2.3，但增加了8.3微升彩信到500微升M9培养基中，用500μl融化的1.5％琼脂糖混合之前，为100毫米的终浓度MS。 （ 注意！MMS是有毒和致突变，且必须带手套，口罩，护目镜，以及白大褂处理）。 </li><li>从垫取下顶部的幻灯片，并添加1微升的浓细胞悬液到垫。通过覆盖垫未使用的烧盖玻片（否1.5厚度，搭配显微镜物镜规范）并按下轻轻地在投影片上的固定化细胞。细胞应在固定的45分钟的琼脂糖凝胶垫干之前进行成像。为了防止在较长的干燥实验，琼脂糖垫可以使用硅密封垫密封。 </li><li>对于DNA损伤的实验中，培养细胞固定在含100毫米彩信20分钟，在湿润的容器中，在室温下成像前的琼脂糖凝胶垫。 </li></ol> 3。准备显微镜数据采集 PALM依赖于检测单个荧光蛋白的精确定位。的敏感性和最佳比对该显微镜是用于数据质量的关键。单分子荧光显微镜通常采用全内反射（TIR）照明通过在薄款盖玻片表面上令人兴奋的只有荧光增强信号噪声比。在这里，成像大肠杆菌内大肠杆菌需要高度倾斜的照明11，它可以在一个TIRF显微镜通过略微降低激发光的角度来实现。 PAmCherry成像更需要一个405 nm的光激活激光和561 nm激发激光。荧光发射被记录在一个电子时产生的114.5毫微米/像素的像素长度的倍率乘以CCD（EMCCD）相机。为了获得最佳的定位精度，像素大小应约匹配的PSF的标准差宽度，以确保充足的采样无需在信号过太多的像素蔓延。 图3显示了一个最小的PALM设置的示意图。 电影1给人的定制显微镜建设进程的印象，见Uphoff 等[7]对仪器的详细说明。 <ol><li>执行常规显微镜对准。测量405 nm和561 nm的连续波激光强度的目标面前。调整561 nm的强度3.5毫瓦（〜400瓦/厘米2）和405 nm的强度10μW（〜1瓦/厘米2）。使用连续可变中性密度滤光片轮，允许从0-10微瓦405 nm的强度逐步调整。关掉激光照射，直到试验开始。 </li><li>将样品在显微镜舞台上，并把细胞变成焦点在透射光显微镜模式（ 图4A）。该EMCCD相机增益已被关闭，以防止损坏相机通过曝光过度。 </li><li>定义一个裁剪视野，以减少数据大小和增加相机读出速度。 </li><li>覆盖了从环境光线和SWIT样本通道上的EMCCD相机增益。 </li><li>帧速率设定为15.26毫秒/帧（包括0.26毫秒相机读出时间）。看到在讨论部分“曝光时间和强度的激励”。 </li><li>显示相机的数据来检查深色背景信号（ 图4B）。 </li><li>开关561 nm激光，并检查激励背景信号（ 图4C）。 </li><li>开关405 nm激光上的POL 1-PAmCherry融合蛋白的光敏化和增加强度，直到荧光的PSF出现。 </li><li>调整激发光束的角度来照射样品接近盖玻片表面的仅一个薄截面。 <ol><li>为此，激光束被聚焦成一个100X NA 1.4物镜的后焦面（ 图3）。平移所述聚焦透镜垂直于光束移动焦点远离物镜使光束以退出下一个角度的目标的中心。 </li><li>嗳米最大化的荧光强度并减少背景信号。需要注意的是严格的TIR激励是最优的内盖玻片表面的100nm的荧光图像，但是，成像DNA结合蛋白与大肠杆菌相关大肠杆菌类核需要更深层次的照明可达0.8微米。 </li></ol></li></ol> 4。数据采集在这里，我们描述的一般协议收购PALM电影。同样的程序适用于成像POL 1-PAmCherry融合蛋白在大肠杆菌完好大肠杆菌细胞在连续的DNA损伤的治疗与彩信。该方法对每个小区不同分子量或拷贝数的融合蛋白的应用将需要不同的采集设置（见讨论部分）。 <ol><li>找到了一个全新的领域细胞在透射光显微镜模式（FOV）和聚焦影像。拿相机的快照记录细胞轮廓（ 图4A）。</li><li>从环境光线覆盖样品并开启EMCCD相机增益。 </li><li>开关561 nm激光，并漂白盖玻片上的细胞自发荧光和背景点为开始数据采集前几秒钟。对于细胞生长和成像中的M9培养基中，并用盖玻片烧通常有很少的荧光背景，但是，预漂白可能是在富生长培养基如LB成像的细胞是有用的。需要注意的是强烈的照明是有毒的细胞，使预漂白应保持在最低限度。 </li><li>开始连续561 nm激发下的收购PALM电影在15.26毫秒/帧。 </li><li>开关405 nm激光，并逐渐增加强度过电影的过程中，达到了高达1瓦/厘米2。避免会引起细胞自体荧光高405 nm的强度。要注意的荧光分子的密度 - 以保持低的激活率是很重要的，这样的PSF是清楚分离出的每个帧（ 图4D-F）。 </li><li>记录万帧/电影（取决于分子的每个单元将被成像的数量）;一个电影通常需要2-3分钟，并且需要0.5-1 GB的硬盘空间取决于视场的大小。 </li><li>重复采集程序的多个视场。请注意，每个视场只能使用一次成像，因为PAmCherry荧光基团得到的光活化漂白和不可逆的。 </li></ol> 5。数据分析一种自动化的和可靠的数据分析框架是用于该方法的性能和效率的关键。我们使用MATLAB编写的定制软件。 <ol><li>使用克罗克等 12描述的算法执行定位分析，霍顿等人 13，HoldenI 等人 14，和维塞尔等人 15的PSF，首先在带通滤波后的图像使用高斯核7像素确定s管径（ 图5A）。候选位置对应的PSF与峰值像素强度高于背景的信号（ 图5B）的4.5倍的标准偏差。每个候选PSF的局部亮的像素用作用于拟合的椭圆高斯函数（ 图5C）的初始猜测。自由拟合参数为：x位置，y位置，X宽度，y轴的宽度，旋转角度，幅度和背景抵消。椭圆高斯掩模时的曝光时间，从而模糊和变形的PSF占分子。 </li><li>从PALM电影到同一视场的透射光显微镜图像的所有帧画出所得的（X，Y）的本地化。 POL 1-PAmCherry的本地化应该出现E的中心区域内大肠杆菌细胞（ 图6A）。如果许多本地化出现细胞外，定位阈值被设置得太低，或者样品包含背景荧光发色粒子。 </li><li>对于自动跟踪分析，MATLAB实现的克罗克等描述的算法的人 12可以使用（见讨论部分“扩散分析”）。出现在随后的帧中的用户定义的跟踪窗口的位置是连接形成一个轨迹。在多个本地化发生在同一个窗口的情况下，磁迹是唯一分配通过最小化步长的总和。对于这些不同的因素的详细讨论，从单粒子跟踪数据计算的扩散系数时，看到维塞尔等人 15 <ol><li>该算法使用一个内存参数，以占轨道在瞬态闪烁或错过了本地化。这里，我们设置内存参数设置为1帧，较高的值可用于跟踪的荧光团与长寿命的暗状态。 </li><li>选择基于以下的校准步骤的合适的跟踪窗口。对于POL 1，我们使用0.57微米（5个像素）。</li><li>运行跟踪算法的一系列跟踪窗参数。计算每个小区测量的轨道作为跟踪窗口，以确定可能的最小跟踪窗口，不分裂的轨道（ 图6B）的函数的数量。 </li><li>积相同的视场的透射光显微镜图像上得到的磁迹进行可视化的细胞内分子运动的空间分布。 POL1曲目应显示扩散的单细胞（ 图6C-D）内局限。 </li><li>如果磁道的一小部分出现细胞间跨越这表明单独的分子被错误地相连，因为跟踪窗口被选择过大和/或光活化率过高（ 图6E）。 </li><li>画出连续的本地化（ 图6F）之间的步长的累积分布。该曲线上升和饱和顺利进行足够大的跟踪窗口但显示了一个隔流板边缘，如果窗口被选择过小。 </li></ol></li><li>分析POL1的扩散特性，计算均方位移（MSD）连续的本地化为每个轨道之间有一个总的N个步骤）： MSD = 1 /（N-1）ΣI = 1，N-1 （X I +1 - X I）2 +（Y i +1的 - Y I）2。 仅包括具有至少4个步骤（N≥5的本地化）跟踪，以减少在MSD值的统计不确定性。 </li><li>剧情MSD值的曲线比通过计算位移在多个帧（ 图6G）一系列的滞后时间。 MSD的曲线的形状可有助于所观察到的分子运动（ 图6H）进行分类。 </li><li>计算每磁道从MSD中的表观扩散系数D *： D * = MSD /（4ΔT） - σ 地名 2 /ΔT。 第二TERM校正估计的定位误差（这里，σLOC = 40纳米并且Δt= 15.26毫秒，看到维塞尔等人 15）。 </li><li>从视场（ 图7A）所有曲目绘制测量D *值的直方图。 </li><li>确定出现势必会根据每个轨道测量D *值的染色体个体POL1分子。分开绑定的人口（锐配电中心在深*〜0 2微米/秒）和自由扩散的分子（广泛分布集中在深* 0.9〜2微米/秒），通过设置一个阈值D * &lt;0.15μm的2 /秒（红柱图7A和7D）。 </li><li>进行定位，跟踪，并在未损坏的细胞（ 图7A-C）和下DNA损伤的治疗与彩信（ 图7D-E）细胞扩散分析POL 1。结合轨道的部分提供了DN的直接定量测量POL 1 在体内的修复活动。 </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+fluorescent+proteins+for+diffraction-limited+and+super-resolution+imaging.">Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging.</a> Trends Cell Biol. (11), 555-565 (2009).</li><li>Elmore, S., M&#252;ller, M., Vischer, N., Odijk, T., Woldringh, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-particle+tracking+of+oriC-GFP+fluorescent+spots+during+chromosome+segregation+in+Escherichia+coli.">Single-particle tracking of oriC-GFP fluorescent spots during chromosome segregation in Escherichia coli.</a> J. Struct. Biol. 151 (3), 275-287 (2005).</li><li>Badrinarayanan, A., Reyes-Lamothe, R., Uphoff, S., Leake, M. C., Sherratt, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+architecture+and+action+of+bacterial+Structural+Maintenance+of+Chromosome+proteins.">In vivo architecture and action of bacterial Structural Maintenance of Chromosome proteins.</a> Science. 338 (6106), 528-531 (2012).</li></ol>

作者宣称他们没有竞争的经济利益。

我们将讨论几个关键因素为实验的成功。 选择与荧光融合蛋白的表达：有光活化和光开关荧光蛋白17的大调色板。具体选择取决于显微镜的特性，特别是激光器和滤波器提供。 405 nm和561 nm处的结合，是理想的通用光激活荧光蛋白。我们选择PAmCherry 6，因为它是单体，并没有表现出聚集的细胞。此外，不可逆的光激活允许计数激活荧光来测量每个细胞蛋白的拷贝数数。代替从质粒表达的融合蛋白的，我们优选的融合蛋白编码基因的染色体插入在野生型基因座。这确保了与荧光已经完全替换所感兴趣的蛋白的rsion和维持野生型表达水平。 光活化率：据以调整光活化率，使得平均每个细胞少于1分子是在荧光状态下在电影的任何帧是很重要的。这依赖于405nm的强度和分子数从左被激活。在非常低的成像密度，然而，并不是所有的分子将会在电影结束前成像或很长的电影有被收购。每部电影记录帧的数量依赖于每个细胞中的融合蛋白和PAmCherry于所使用的激发条件下的平均光漂白寿命的拷贝数。 POL1的拷贝数为〜400个分子/细胞1和指数漂白寿命分布的平均值为〜4帧。通过逐渐增加405nm处的强度，激活均匀分布在电影的10,000帧。因此，每个小区是职业医学共计〜1,600帧的灭蝇灯用荧光分子，确保专业服务公司和电影中的10,000帧的跟踪并发症几乎没有重叠。 曝光时间和强度的激发：最重要的，曝光时间必须足够短，观察锐利的PSF几乎没有运动模糊。然而，帧速率的选择应以产生超出定位不确定性连续帧之间可观察到的分子运动，否则至关重要的光子被过采样的轨道浪费。未结合的分子的运动必须在足够长的时间间隔进行采样是从由于本地化的不确定性结合分子的视运动清晰可辨。当曝光时间被设置，PSF强度应进行调整。 PSF中的定位精度随检测到超过一帧的持续时间的光子数。较高的激发强度增加光子发射率BU吨也光漂白率和背景信号。用最低的激发强度，给出所需的定位精度。对于POL 1-PAmCherry我们选择15.26毫秒/帧和3.5毫瓦561 nm激发（400瓦/厘米2）。它确认细胞存活率为特定的成像条件通过监测细胞的生长和形态之前和之后的数据采集（见补充信息中Uphoff 等 7）是重要的。 POL1展品〜2秒的时间绑定在体内 7跳空DNA底物，因此，我们预期大部分分子要么在绑定和非绑定状态，轨道的整个期间。结合分子出现基本上不动，因为染色体位点有一个扩散系数的幅值低几个数量级（〜10 -5μm的2 /秒，埃尔莫尔等人 18），比在细胞质中POL1扩散（〜1微米2 </s向上&gt; /秒）。 扩散分析：表观扩散系数D *是从各个轨道的MSD计算，平均超过一个最小的4个步骤（5个帧），以减少统计误差。需要注意的是分子〜75％的范围内小于5帧漂白为所述成像条件。如此短的轨道没有提供足够的统计确定性区分绑定和未绑定的分子。然而，绑定和未绑定分子的相对分数对蛋白质的活动，报告是独立的分析曲目总数。 因为不确定性增加了一个明显的随机步的分子15的每个定位是非常有用的占PSF定位误差（σ 同上 ）中的D *的计算。 为了提高约束和扩散的分子分类，我们建议计算D *机器人h，从单步位移和位移超过两帧的时间。然后，可以设置两个独立的D *阈值：D *（15毫秒）&lt;0.15微米2 /秒和D *（30毫秒）&lt;0.075微米2 /秒。 注意，D *]是受轨道和运动过程中的曝光时间所造成的模糊扩散的细胞隔离的表观扩散系数。来提取精确的中立扩散系数，它已被证明有用观察到的运动进行比较的基础上随机布朗运动模型5,7的模拟数据。模拟数据，也可以用来测试数据分析程序。 这种方法的潜在应用：我们描述了在结合到染色体可视化和量化的体内蛋白-DNA相互作用通过在蛋白质的迁移率的变化的一般方法。的DNA或活动参与修复的RNA结合蛋白，复制，转录，和染色体维持由此可以随后在实时在与下面的光学衍射极限的空间分辨率的单个细胞水平。光活化的单分子跟踪扩展被限制在每个小区的几个标记分子传统的跟踪方法。测量体内分子扩散的一种替代方法是荧光漂白恢复（FRAP）之后。而FRAP是用于测量大细胞全球扩散特性非常有用的，它在其解决几个分子种类具有不同的迁移率在空间异构环境中，尤其是对于小的细菌细胞的能力是有限的。 本公司已申请光敏单分子跟踪测量DNA结合活性和在大肠杆菌中的各种不同的蛋白质的亚细胞定位大肠杆菌包括POL 1，DNA连接酶，FIS蛋白质，DNA聚合酶III 7，以及染色体蛋白质MukB，E和F 19的结构维修。我们预期该方法还可以适用于其他类型的细胞。

这个协议描述了直接测量在活的大肠杆菌细胞蛋白-DNA相互作用的。该技术利用了单个荧光标记的蛋白质的扩散系数的变化，因为它结合了染色体（ 图1）。以证明我们用DNA聚合酶填充DNA的缺口随链复制和切除修复途径1 I（POL 1），一个典型的DNA结合蛋白的方法。 超分辨率荧光显微镜的问世使分子结构的可视化的细胞与纳米级分辨率。光活化定位显微镜（PALM）采用荧光蛋白质，可以从初始暗状态被激活以荧光的状态（ 图2）。只有所有的标记分子的一个子集在任何时间被启动，以确定独立吨它们的位置以顺序的方式，他总标记分子的浓度样品2中。每分子的定位精度主要依赖于荧光点扩散函数（PSF）的大小，收集光子的数目，并且背景信号3。这种方法的许多应用集中在蜂窝式结构的改进的可视化。那一掌可与单分子组合跟踪的实现4开辟了新的途径，直接跟在活细胞中任意数字标记的蛋白的运动。更高的灵敏度和荧光显微镜的时间分辨率现在允许单个扩散荧光蛋白跟踪在细菌胞质5。 在这里，我们采用PAmCherry，一个精心设计的荧光蛋白不可逆转换从最初的非荧光状态下照射时荧光状态，405纳米的光6。激活PAmCherry荧光团可以是图像D由激发在561 nm和跟踪几帧，直到漂白。我们展示了使用融合POL1和PAmCherry来识别单个蛋白质的瞬态DNA结合事件的方法的能力。细胞的甲基磺酸甲酯（MMS）的治疗会导致由碱基切除修复酶变成跳空DNA底物DNA甲基化损伤。我们的方法显示效果清晰单一POL 1分子的结合响应彩信损坏7。

<table border="0" cellpadding="0" cellspacing="0" width="1444">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">E.coli strain carrying a chromosomal insertion for a PAmCherry DNA-binding fusion protein</td>
			<td style="width:187px;"></td>
			<td style="width:477px;"></td>
			<td style="width:467px;">created by Lambda-Red recombination</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM amino acids</td>
			<td>Invitrogen</td>
			<td>11130-051</td>
			<td>minimal medium supplement</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM vitamins</td>
			<td>Invitrogen</td>
			<td>11120-052</td>
			<td>minimal medium supplement</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose</td>
			<td>BioRad</td>
			<td>161-3100</td>
			<td>certified molecular biology grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope coverslips No 1.5 thickness</td>
			<td>Menzel</td>
			<td>BB024060SC</td>
			<td style="width:467px;">remove background particles by heating slides in furnace at 500 &#176;C for 1h</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methyl methanesulfonate (MMS)</td>
			<td>Sigma-Aldrich</td>
			<td>129925</td>
			<td>CAUTION: toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PALM setup</td>
			<td>home-built</td>
			<td></td>
			<td>described in Uphoff et al. 2013</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td>for data analysis and visualization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Localization software</td>
			<td style="width:187px;">custom-written, available online</td>
			<td>http://www.physics.ox.ac.uk/Users/kapanidis/Group/Main.Software.html</td>
			<td style="width:467px;">MATLAB and C++ software package that can be adapted for localization analysis. Cite Holden et al. 2010 if used in publication.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tracking software</td>
			<td>available online</td>
			<td><a href="http://physics.georgetown.edu/matlab/">http://physics.georgetown.edu/matlab/</a></td>
			<td style="width:467px;">MATLAB implementation by Blair and Dufresne. Cite Crocker &#38; Grier 1996 if used in publication.</td>
		</tr>
	</tbody>
</table>

visualizing protein-dna interactions in live bacterial cells using photoactivated single-molecule tracking

蛋白质-DNA相互作用是在许多基本的细胞过程的心脏。例如，DNA复制，转录，修复和染色体组织是由能够识别特定的DNA结构或序列的DNA结合蛋白管辖。 体外实验已经帮助生成详细的模型对许多类型的DNA结合蛋白的功能，但，这些方法和它们的组织在活细胞的复杂环境的确切机制仍然远远低于理解。我们最近推出了使用光活化定位显微镜（PALM）结合单分子跟踪量化活的大肠杆菌细胞DNA修复活动的方法。我们一般的做法通过在与染色体的关联在一个单一的蛋白质的流动性的变化识别个人DNA结合事件。结合分子的比例为蛋白质行为的直接定量测量ivity和丰度底物或结合位点在单细胞水平。在这里，我们描述了该方法的概念，并展示样品制备，数据采集和数据分析程序。

光活化的单分子跟踪研究体内蛋白质-DNA相互作用的概念图示于图1。 PAmCherry融合蛋白在大肠杆菌活检测大肠杆菌细胞中以连续的方式通过随机光活化的单分子具有405nm的光在每个细胞少于一个分子在一个时间的频率。活化分子连续561 nm激发下成像。在细胞中的分子运动可以通过连接附近的本地化在一系列的帧，直到不可逆漂白被跟踪。因为DNA结合蛋白的扩散结合后的染色体减慢，表观扩散系数D *每磁道获得的直接报告关于个别蛋白质-DNA相互作用。 图2演示了POL 1-PAmCherry融合蛋白的光活化的活大肠杆菌大肠杆菌细胞。在405 nm处的强度邻的影响Ñ ​​荧光分子的密度可以看出，在图4中 。注意的是，密度是不完全由405nm的强度，但另外由分子可用于激活的数目来确定;其余分子的池耗尽超过PALM电影的过程。 进行定位分析，对于Palm电影如图5每一帧。我们用动分子在活细胞固定细胞或分子结合的测量定位精度。我们收购的设置给了σ 禄 = 40纳米的定位精度，与理论预测3协议。 所得POL1本地化占据单元格的中央区域（ 图6A），从广义扼要的E的空间组织大肠杆菌类核7。多数POL1在完好的细胞追踪显示DIF如图6C融合。一个典型的单元格中包含几百个POL 1曲目（ 图6D），以每E.约400 POL 1分子的拷贝数一致大肠杆菌细胞1 图6B和6E-F提供的指导选择合适的跟踪窗口参数-如果跟踪窗口太大时，不同的分子更可能成为错误地连接到一个轨道，如果该跟踪窗口太小时，轨道与步长将被分割。 MSD的曲线POL1短的时间滞后和在饱和由于细胞禁闭（ 图6G）较长的滞后时间线性上升。不同类型的分子的运动可以通过MSD分析来识别。定向运动给出的抛物线曲线;布朗运动，其特征在于用直线;密闭扩散曲线达到平稳;一对MSD曲线为不动的微粒的偏移量表示的升ocalization不确定性（ 图6H）。对单粒子跟踪和故障排除提示其他信息可以在阿尔诺等 16发现我们先前采用的方法来测量响应于外源DNA烷基化损伤7 POL1的DNA修复活性。 POL 1的D *直方图跟踪在完好的细胞显示了扩散的分子（ 图7A-C）的优势种群。 2.7％的结合POL 1分子的一小部分可能参与了滞后链复制和修复内源性DNA损伤。在连续100毫米彩信损坏，用D *〜0 2微米/秒提高到13.8％（ 图7D）轨道的人口。这些曲目代表进行DNA修复合成，以填补单核苷酸缺口的碱基切除修复途径的一部分，个人POL1分子。结合的磁道的位置显示单独的位置DNA损伤与修复部位（ 图7E-F）。 <img alt="图1" fo:content-width="2.5in" src="/files/ftp_upload/51177/51177fig1highres.jpg" width="300" /> 图1。该方法的图形表示（A）的荧光蛋白PAmCherry可以从最初的非荧光状态照射后光活化与405纳米的光。明亮的状态是兴奋，561 nm和发射大约600nm的荧光，直到荧光漂白剂不可逆的。 （b）控制光活化率允许成像只有每个细胞一个随机激活PAmCherry融合蛋白在任何时间，而尚未在黑暗状态下启动或已经漂白遗骸分子的任意大的游泳池。 （C） 的荧光分子的位置是从分离的PSF和TR的中心确定在得到确认为几个帧，直到光漂白。许多分子的（D）的轨道被记录在一个连续的方式。 （EF）与染色体靶序列或结构的DNA结合蛋白的相互作用将暂停随机扩散运动。结合和未结合的分子是由表观扩散系数D *从单一磁迹中提取区分。结合分子所产生的部分给出了定量测量体内的DNA结合蛋白的活性。 <a href="https://www.jove.com/files/ftp_upload/51177/51177fig1highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51177/51177fig2highres.jpg" width="600" /> 图2。 POL 1-PAmCherry在现场E.光敏大肠杆菌细胞 。比例尺：1微米。原理图中显示的每个面板下方。 （<html> A）传输的细胞固定在琼脂糖垫的光学显微镜图像。 （B）Phototactivating在一个小区中的单个PAmCherry荧光团。 （ 三）较高的光敏化率提高了荧光分子的数量。 （ 四）从Palm电影综合PAmCherry荧光。 <a href="https://www.jove.com/files/ftp_upload/51177/51177fig2highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图3" fo:content-width="4in" src="/files/ftp_upload/51177/51177fig3highres.jpg" width="400" /> 图3。最小PALM设置的光激活与成像PAmCherry融合蛋白的示意图D1：分色镜（ 如 550纳米长通）。 D2：分色镜（ 如 570纳米长通）。 L1：准直透镜。 L2：TIR透镜。 L3：管镜头。1177/51177fig3highres.jpg“目标=”_blank“&gt;点击这里查看大图。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51177/51177fig4highres.jpg" width="500" /> 图4。代表图像从Palm电影与15.26毫秒/帧比例尺：1微米。 （ 一 ）传染的细胞固定在琼脂糖垫的光图像。 （B）该EMCCD相机与激光测得的暗背景图像关掉。 （三）激励的背景光激活之前，连续561 nm激发下的图像。 （DF）增加405 nm的强度导致PAmCherry更高的光敏化率，连续561 nm激发下成像。盒装区域如下所示放大。 （D）低405nm的强度（&lt;1μW）活性成分每FOV很少的荧光分子。 （E）中405nm的强度（〜2微瓦）光活化结果在一个良好的PSF密度定位与跟踪分析。 （F）的405 nm的强度（〜10μW）激活多个荧光分子在某些细胞中，它掩盖了定位和跟踪分析。 <a href="https://www.jove.com/files/ftp_upload/51177/51177fig4highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51177/51177fig5highres.jpg" width="500" /> 图5。插图定位分析。比例尺：1微米 （ 一 ）带通滤波消除杂散像素噪声和平坦整个视场的强度梯度。 （B）候选的PSF进行标识，根据该被选择，以尽量减少假阳性和假阴性检测的用户定义的阈值的滤波图像。该阈值分割，LD对应于候选象素的由背景的标准偏差（信号 - 噪声比，信噪比）划分的最小强度。 （ 三）局部亮的像素是通过阈值作为初始定位的猜测（橙色交叉）为二维椭圆高斯拟合。比例尺：0.5微米。由此产生的超分辨率本地化（蓝十字）具有σ 禄的平均精度= 40纳米。 <a href="https://www.jove.com/files/ftp_upload/51177/51177fig5highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51177/51177fig6highres.jpg" width="500" /> 。图6插图跟踪分析比例尺：1微米。 （ 一 ）POL 1-PAmCherry的一个例子细胞检测到的所有本地化。轨道（B）号检测版中的示例小区作为跟踪窗口的功能。小的跟踪窗口拆分分子的轨迹，从而导致伪迹大量曲目。虚线表示我们选择跟踪窗口参数（0.57微米，5个像素） - 这给检测步骤充分散发，保持不同分子的运动轨迹完好之间的良好折衷。 （三）示例轨道单POL 1-PAmCherry分子。 （四）随机颜色显示的所有测量轨道。 （E）的跟踪工件如果选择了跟踪窗口过大（此处为0.8μm，7个像素）或每帧的点扩展函数的密度过高。步长为跟踪窗口（F）的累积分布：0.34微米（3像素，红色线），0.57微米（5像素，蓝色线），和0.80微米（7像素，绿线）。请注意，0.34微米跟踪窗切断步骤比0.34微米长，清楚截断的步骤的全面分发。在0.57微米跟踪窗口检测步骤几乎相同的分布一样的0.80微米跟踪窗口。 （G）的MSD曲线显示POL1的局限扩散。 （H）示意图MSD曲线定向运动，布朗运动，密闭扩散和不动的颗粒。 <a href="https://www.jove.com/files/ftp_upload/51177/51177fig6highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51177/51177fig7highres.jpg" width="500" /> 图7。 POL1在现场大肠杆菌的DNA修复活性的直接测量大肠杆菌细胞比例尺：1微米。 （ 一 ）直方图的表观扩散系数D *用于在未受损细胞的视场（N = 4,162首曲目）4个或更多步骤中的所有曲目。归类为b个分子群体<html> OUND以红色突出显示。 POL 1-PAmCherry的（BC）专辑曲目显示在透射光显微镜图像。分类为根据其扩散系数的约束轨迹显示为红色。 （D）D *直方图中的细胞成像（N = 2128磁迹）之前固定在琼脂糖垫用100mM MMS和孵育20分钟测量POL1轨道。从事DNA修复分子结合的人口以红色显示。 （EF）POL 1-PAmCherry轨道上的透射光显微镜图像显示为红色单POL1 DNA修复活动的轨迹。 <a href="https://www.jove.com/files/ftp_upload/51177/51177fig7highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="视频1" fo:content-width="4in" src="/files/ftp_upload/51177/video.jpg" width="400" /> 电影1。构建一个定制的PALM设置 。JoVE_Uphoff_Movie1.avi“目标=”_blank“&gt;点击此处查看视频。

Watch this Scientific Journal Video about Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking at JoVE.com

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

光敏定位显微镜（PALM）结合单分子跟踪允许直接观察活的大肠杆菌细胞蛋白-DNA相互作用的定性和定量。

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

visualizing-protein-dna-interactions-live-bacterial-cells-using

Research

JoVE Journal

Immunology and Infection

17.6K 次观看.  University of Oxford. 光敏定位显微镜（PALM）结合单分子跟踪允许直接观察活的大肠杆菌细胞蛋白-DNA相互作用的定性和定量。 

视频: Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

科研

免疫与感染

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

评估气孔反应活细菌细胞，采用全叶成像

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

成像InlC的分泌由细菌病原体调查感染蜂窝<em&gt;单核细胞增生李斯特氏菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

使用madSTORM进行高分辨率，超分辨率的T细胞成像

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

利用共焦显微镜可视化巨噬细胞胞外陷阱

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...