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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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><tbody><tr><td>&lt;em&gt;E. coli&lt;/em&gt; strain carrying a chromosomal insertion for a PAmCherry DNA-binding fusion protein</td><td></td><td></td><td>created by Lambda-Red recombination</td></tr><tr><td>MEM amino acids</td><td>Invitrogen</td><td>11130-051</td><td>minimal medium supplement</td></tr><tr><td>MEM vitamins</td><td>Invitrogen</td><td>11120-052</td><td>minimal medium supplement</td></tr><tr><td>Agarose</td><td>BioRad</td><td>161-3100</td><td>certified molecular biology grade</td></tr><tr><td>Microscope coverslips No 1.5 thickness</td><td>Menzel</td><td>BB024060SC</td><td>remove background particles by heating slides in furnace at 500 &amp;deg;C for 1 hr</td></tr><tr><td>Methyl methanesulfonate (MMS)</td><td>Sigma-Aldrich</td><td>129925</td><td>CAUTION: toxic</td></tr><tr><td>PALM setup</td><td>home-built</td><td></td><td>described in Uphoff &lt;em&gt;et al.&lt;/em&gt;&lt;sup&gt;7&lt;/sup&gt;</td></tr><tr><td>MATLAB</td><td>MathWorks</td><td></td><td>for data analysis and visualization</td></tr><tr><td>Localization software</td><td>custom-written, available online</td><td>http://www.physics.ox.ac.uk/Users/kapanidis/Group/Main.Software.html</td><td>MATLAB and C++ software package that can be adapted for localization analysis. Cite Holden &lt;em&gt;et al.&lt;/em&gt;&lt;sup&gt;13&lt;/sup&gt; if used in publication.</td></tr><tr><td>Tracking software</td><td>available online</td><td>&lt;a href=&quot;http://physics.georgetown.edu/matlab/&quot;&gt;http://physics.georgetown.edu/matlab/&lt;/a&gt;</td><td>MATLAB implementation by Blair and Dufresne. Cite Crocker &amp;amp; Grier&lt;sup&gt;12&lt;/sup&gt; 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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

17.7K 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

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

科研

生物化学

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

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

单细胞物种间细菌相互作用的动力学可视化

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

免疫与感染

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 ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

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

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

通过电穿孔内在和生物分子荧光观察生活中的微生物

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

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb &#955; DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified &#955; DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified &#955; DNA substrate and a Quantum-dot labeled protein.

Qdot 标记蛋白与现场特异修饰的λ DNA 在单分子水平上的相互作用可视化

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. ...

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.

活细菌中蛋白质-蛋白质相互作用的FLIM-FRET测量。

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster ...

基于活细胞单分子成像的蛋白质相互作用定量

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

作者聚焦:活人细胞中蛋白质-凝聚物动力学的评估

Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells.

生物分子缩合物中蛋白质相互作用动力学的单分子测量

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number ...

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

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此视频中的章节

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

可视化单分子络合物在体内使用高级荧光显微

单细胞物种间细菌相互作用的动力学可视化

结合单分子操纵和成像蛋白-DNA相互作用的研究

通过电穿孔内在和生物分子荧光观察生活中的微生物

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

Qdot 标记蛋白与现场特异修饰的λ DNA 在单分子水平上的相互作用可视化

活细菌中蛋白质-蛋白质相互作用的FLIM-FRET测量。

生物分子缩合物中蛋白质相互作用动力学的单分子测量

生物分子缩合物中蛋白质相互作用动力学的单分子测量