这项工作是由马克斯 - 普朗克协会。

1。样品制备<ol class="lalpha"><li>准备盖玻片 盖玻片应清洗尘粒的TIRF从盖玻片（ 图1A，电影1）产生的背景信号是敏感。 <ol><li>在有盖的陶瓷人用钳子盖玻片到位。 </li><li> 1 M氢氧化钠（可重复使用多次）填补玻璃容器。 </li><li>缓慢连续旋转（轨道摇床）下孵育2小时。过多的潜伏期（&gt; 8小时），将创建不透明的盖玻片。 </li><li>下缓慢连续旋转5分钟，用蒸馏水清洗两次。 </li><li>储存在陶瓷支架覆盖100％的乙醇。 </li></ol></li><li> 枯草芽孢杆菌细胞准备 <ol><li> 0.01外径600 - 0.05稀释至5毫升的适度增长，媒体和成长指数阶段（0.3-0.7 OD 600）。 </li><li>准备在1.25％琼脂糖水或中等。使用合成纤维媒体减少背景荧光。溶于1.5毫升塑料管粉在95℃，加热块存储在72°C。 </li><li>琼脂糖小滴添加到幻灯片和记者到了第二张幻灯片的平垫中间。至少2分钟后，或前仔细单独的幻灯片使用。 </li><li>微型离心机在降速为1分12.000转500μL细胞。 </li><li>弃上清，重悬于50μL介质中的颗粒。 </li><li> 2-4μL的细胞到琼脂糖垫的中心。 </li><li>采取用钳子从乙醇装满容器的清洗盖玻片，用加压空气干燥，小心地放在样品。 </li><li>让细胞定居至少2分钟前显微镜。 </li><li> VALAP（凡士林，羊毛脂和石蜡，在​​同等重量下热混合密封盖玻片的边缘，适用于小刷子或吐口水ULA）长期成像。 </li></ol></li><li> 酿酒酵母细胞的准备 <ol><li>稀释前培养和成长指数阶段（0.2-0.8外径600）至少5个小时，在适当的媒体。 </li><li>采取盖玻片，镊子和干燥的压缩空气从容器。 </li><li>传播5μL刀豆蛋白A盖用吸管和加压空气干燥防滑的解决方案（刀豆，2微克/毫升蒸馏水）。刀豆结合酵母细胞壁的碳水化合物，酵母细胞无法移动。 </li><li> 4.5μL酵母细胞悬液转移以刀豆涂层盖玻片的一面。小心地将盖玻片，在显微镜幻灯片。一个边缘开始，让缓慢下降，以避免形成气泡。 </li><li>让细胞定居至少2分钟前显微镜。 </li><li>密封边盖玻片与VALAP（见上文）长期成像。 </li></ol></li></ol><p=“jove_title”的2课。图像采集<ol class="lalpha"><li>显微镜的设置 我们完全的自动化iMIC立场与奥林巴斯100X 1.45 NA目标（至光电）建立一个定制的TIRF进行所有实验。 75兆瓦，在488纳米（相干蓝宝石）和561纳米（Cobolt牛仔）DPSS激光器作为光源。激光器通过AOTF的选择，并通过宽带光纤到iMIC执导。一个振驱动两轴扫描头被用于漂白（FRAP还原力）的位置，以调整后的入射角或荧光恢复。额外振被用于萤光，FRAP还原力和的TIRF之间切换。图像采集与安道尔IXON DU-897 EMCCD相机，在镜头前的2倍放大倍率镜头。收购控制的实时采集（截至光电）软件包。 应考虑以下几点： <ol><li>可编程控制照明光源（激光升INE）和角度的TIRF是必不可少的重复性的结果，尤其是多色彩的TIRF，需要调整，以确保每个激发波长等于渗透渐逝波的入射角。在我们设置的TIRF角度可以调整，瞬间通过两个电流计，这是直接从现场采集软件包控制。 </li><li>此外我们的自定义安装允许通过第三振萤光，TIRF和FRAP之间的快速切换，允许简单的测量定位动力学的TIRF追踪的对象。 </li><li>目的型的TIRF需要高数值孔径的NA&gt; 1.4。我们使用奥林巴斯100X NA 1.45目标。 </li><li> EMMCD相机很好地工作的TIRF由于其高信噪比。允许最佳灵敏度的成像，我们采用了512x512像素的EMCCD相机背面照亮了16微米的像素尺寸。为了保持最高分辨率，我们放置了一个2X MAGNification镜头在镜头前。 </li><li>随着的TIRF是一种广角技术，激光束的强度被传播出去。因此，强大的激光器往往提高成像。我们用75兆瓦二极管泵浦固态（DPSS）激光器在488 nm和561 nm处。 </li><li>为了保持最佳生长条件下的成像过程中的细胞，用适当的环境控制。我们使用了一个定制的加热室（图1B）研究酵母温度敏感突变体。对于中型交流，在收购过程中，我们用简单的流室或玻璃底培养皿倒置显微镜可以很容易地从上述访问。 </li></ol></li><li>调整参数 <ol><li>使用亮场照明的细胞识别的位置。红色发光二极管是特别好，因为它们不引起显着的光损伤。 </li><li>切换到激光照排，并选择适当的激光器和滤波器的组合激发的荧光选择。确保的TIRF角是亚临界的，否则你将无法检测到任何信号，GFP融合蛋白的产生。如果设置有疑问的TIRF角度为0°，以获得一个垂直激光束。找到（方面临的盖玻片的细胞）的细胞的顶端部分。改变激光束的入射角，直到信号消失，然后慢慢增加，直到点的角度，在荧光在细胞表面再现（ 电影）。调整ž再次集中在表面的最佳位置。可接受的TIRF角度创建没有模糊的光环边缘的细胞（ 图1C）。如果在安装过程中发生显著照片漂白，保存的TIRF角度和移动开始收购前，本色的舞台上的地位。 </li><li>调整激光强度和摄像机的增益最大化动态范围（高信号噪声比不饱和像素）。通常EMCCD相机是最优的检测非常低的信号，高信号噪声大鼠IOS。普通的CCD相机为更强的信号产生低噪音，并提供了更宽的动态范围。 </li><li> TIRF显微镜是非常敏感的小盖玻片的高度差异，导致只有少数细胞，它可以在同一时间（ 图1D）成像。对于小细胞采集区域（ROI）的地区的利益，因此经常被减少到分区域，包含选择的对象。这也减少了从芯片到硬盘驱动器的数据，往往限速在高帧频一步转移所需的时间。 </li><li>为双色的TIRF实验，一定要尽量减少出血，通过荧光。对于RFP / GFP的对使用利福平单独的排放过滤器是弱兴奋488纳米的光（ 图2A）。大多数过滤器彼此不完全一致。过滤偏移可以通过使用混合细胞中的荧光珠纠正。为了实现可比性的穿透深度，调整的TIRF角度分别FOR每个激光线。一个典型的工作流程如图所示。 2B。 </li><li>影响图像质量的一个关键参数是激光准直和一个干净的目标。对齐的激光，用于TIRF成像的Z-位置上的第一焦点。然后取出样品，并清理所有石油的目标（剩余油会导致激光散斑衍射光束轮廓）。投射在天花板上的TIRF模式的激光校准激光是在与目标（使用参考点，始终佩戴激光护目镜，避免所有的反射光路）直线0°位置。激光光斑聚焦到最小尺寸。我们的设置提供了一个可移动的镜头，振镜和显微镜之间的位置，以方便快速校准。激光轮廓优化调整后，应形成良好的定义大致圆形的现场。每一次会议之前执行校准，以获得最佳的图像质量。 </li></ol></li></ol> 3。代表结果<ol class="lalpha"><li> 肌动蛋白的同源性和细胞壁的酶在枯草芽孢杆菌的分布。我们拍摄细胞表达的肌动蛋白的同源性MBL或transpeptidase PbpH的TIRF和定期萤光的绿色荧光蛋白融合。中的TIRF穿透深度明显减少限于面临盖玻片表面（ 图3A，B）的。弱表达的的transpeptidase PbpH定位皮质补丁，几乎是由萤光可见，但可以清楚地区别的TIRF（ 图3B）。减少渗透可以观察PbpHGFP信号隔，萤光线，但出现的TIRF点，箭头 ）（ 图3B。 </li><li> 蛋白质结构域在酵母细胞膜的质膜蛋白质在酵母中形成网络状的图案。图。 1C和2。除了这些网络状的图案，使明确区分的TIRF改进对比度，有时可以被检测到的微弱信号，完全由TIRF显微镜。作为例子，我们拍摄了酿酒酵母的TORC2从复杂的组件Bit61 GFP融合。这种蛋白是弱的表达，并形成小的皮质补丁，只有少数蛋白的副本，每补丁9。在萤光只有几片以上的强大背景是可见的，而补丁可以很好的信号噪声比的TIRF（ 图3C）成像。因此特别适合的TIRF研究弱荧光的结构和蛋白质在细胞表面。高轴向分辨率的TIRF提供已经减少背景荧光。进一步改善图像对比度，可通过各种图像处理步骤。一个简单的标准技术，是当地的背景均衡，从一个模糊的形象减去原。模糊可进行各种低通滤波器，包括中位数或高斯滤波器（ 图3D）。当地背景减法消除噪声，加剧了高强度的结构。然而，这往往在亏损的精细结构和高强度地区的夸张放大，箭头 ）（ 图3D价格。一个卓越的过程是二维卷积，ImageJ的，Matlab或惠更斯（科学卷成像BV），如免费或市售的软件包，可以执行。的TIRF图像提供了很好的轴向分辨率，因此近二维点扩散函数（PSF）。我们实验确定由40纳米荧光珠获得相同的设置与实际成像（目标，相机，激发和发射波长）用于实验涤纶短纤，然后用一个经典的最大似然算法的卷积图像涤纶短纤惠更斯。时间电影（ 电影3和4）的GFPRas2，或Fet3GFP和Pma1RFP失效的后卷积对比增加证明。双彩色图像的关键是最大限度地对比，能够量化的共存值（ 电影4）。 </li><li> 分析蛋白质在细胞皮层的营业额。的TIRF和FRAP皮层蛋白质的动力学研究提供强大的组合。然而，为了使这些技术的使用效率，迅速控制激光的角度和立场是必需的。中的TIRF，宽领域的技术，激光需要的后焦平面上的集中和需要遵循一个浅的入射角，以实现所需的反射。相比之下，FRAP还原需要集中直接对样品定位和空间精度高，允许定义的结构或地区的漂白激光。从光电子直到我们在我们的实验中使用的设置，提供了非常快速和直观的控制超过日ESE参数。两个控制振镜是用来调整FRAP还原位置在X，Y或设置的TIRF入射角。第三个镜像的TIRF和FRAP照明（激光扩大的TIRF路径的额外的镜头）使用单独的光束路径之间切换。所有设置控制由洛杉矶（现场采集）软件，可迅速为每一个实验，以达到最佳的条件进行校准。该软件还允许在图像采集，这是必不可少的能动结构FRAP还原实验的漂白职位的定义。 使用此功能，我们可以排除MBL细丝treadmilling作为MBL含补丁观测运动（ 图3E）的来源。这个观察促使我们寻找替代动力机制，并最终导致我们的活力，细胞内蛋白质复合物（包括MBL）移动通过活动完全出乎意料的类型识别细胞壁的TY合成酶居住在细胞外4。以类似的方式，我们能够观察到酵母质膜蛋白质的缓慢的重排，分发到网络域覆盖整个细胞表面（ 图3F）般。 这些例子说明如何动态和皮层蛋白质的图案可以直接结合的TIRF和FRAP技术评估。 </li></ol></SPAN&gt; 4。代表结果<img src="/files/ftp_upload/3982/3982fig1.jpg" alt="图1"/> 图1。调整参数。肮脏与清洁的合作verslip。从盖玻片上的灰尘产生的背景信号干扰与GFP / RFP一个潜在的信号，清洗盖玻片有较少的背景。B.定制电加热控制器设定的温度（A），温度探头（b）和样品架（C）随入射角（从顶部从左上到右下）。C.酵母蛋白Pma1GFP的的成像。图像显示在临界角突然失去信号和结构信息和信噪比逐步减少。D.不均匀电场中。密集的荧光珠悬浮成像，显示，视野只有部分的重点是。比例尺在A，B：2微米，在C：10微米。 <img src="/files/ftp_upload/3982/3982fig2.jpg" alt="图2"/> 图2。双色成像。 A.出血通过Pil1RFP。 Pil1RFP兴奋488 nm和561 nm激光和荧光录得双带通滤波器。弱信号的Pil1RFP是在GFP通道可见。比例尺2微米B.典型步骤，以避免流血，通过双彩色成像。单独的过滤器集的细胞成像后，他们deconvolved和对齐（使用荧光珠）前被合并分析。 <img src="/files/ftp_upload/3982/3982fig3.jpg" alt="图3"/> 图3。代表结果。 A. 在 B GFPMbl分布比较枯草使用的TIRF或定期萤光显微镜（EPI）。蓝色：从明场图像的细胞边界。图像代表一个时间序列的平均预测表明监测不同的成像模式的能动MreB补丁覆盖面积。B.改善轴向分辨率和对比度图像的TIRF猝然transpeptidase PbpHGFP 在 B 枯草。箭头指示出现在萤光环的TIRF补丁的间隔染色。曝光时间：100毫秒。C.萤光和的TIRF照明猛龙复杂的组件在S 酵母 Bit61之间的比较。 bit61是非常弱表达。曝光时间：250毫秒D.不同的图像处理方法的比较。系列显示原料Pma1GFP的TIRF形象，高斯模糊图像中位数，以及卷积减法惠更斯在使用最大似然算法。E.的TIRF-FRAP还原单一GFPMbl补丁跨越一个B.移动（以上星号） 枯草细胞。非恢复补丁和强度分布沿kymograph（虚线）出的议案来源treadmilling规则kymograph。 楼的TIRF FRAP还原力的酵母表达Pma1GFP的细胞。请注意的扩散闭幕在皮质的染色模式的差距。比例尺：2微米。在几秒钟的时间。 电影1电影显示B。枯草细胞表达RFP融合蛋白的一个肮脏的盖玻片上。注意RFP的信号从背景噪音是很难区分的。周期时间100毫秒。 <a href="http://www.jove.com/files/ftp_upload/3982/3982_Movie1.mp4" target="_blank">点击这里观看电影</a> 。 电影2电影与不同的入射角Pma1GFP。角度改变了从亚临界到临界角，直到信号丢失。请注意，是在亚临界角的内部信号可见，造成噪声图像，直到在临界角，只是在下午的蛋白质是可见的。周期时间200毫秒。比例尺：2微米。 <a href="http://www.jove.com/files/ftp_upload/3982/3982_Movie2.mov" target="_blank">点击这里观看电影</a> 。 电影3电影酵母GFPRas2（显示alternat）ING RAW和deconvolved的TIRF图像。 GFPRas2是一个快速移动的蛋白质（T1的未公布的数据2S / 2），快速采集时间是很重要的，解决的动态行为。周期时间150毫秒。比例尺：2微米。 <a href="http://www.jove.com/files/ftp_upload/3982/3982_Movie3.mov" target="_blank">点击这里观看电影</a> 。 电影4。双色酵母蛋白Fet3GFP和Pma1RFP的电影。首先代表10帧RAW图像和最后帧deconvolved。这部电影显示的图像处理分析共存的重要性。 RAW图像的模糊变得尖锐，使分析成为可能。循环时间5秒。比例尺：2微米。 <a href="http://www.jove.com/files/ftp_upload/3982/3982_Movie4.mov" target="_blank">点击这里观看电影</a> 。

<ol>
	<li>Axelrod, D., Thompson, N. L., Burghardt, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+inflection+fluorescent+microscopy.">Total internal inflection fluorescent microscopy.</a> J. Microsc. 129, 19-28 (1983).</li><li>Axelrod, D., Omann, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+microscopy.">Combinatorial microscopy.</a> Nat. Rev. Mol. Cell Biol. 7, 944-952 (2006).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+surface+fluorescence+with+very+high+aperture+microscope+objectives.">Selective imaging of surface fluorescence with very high aperture microscope objectives.</a> J. Biomed. Opt. 6, 6-13 (2001).</li><li>Dominguez-Escobar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processive+movement+of+MreB-associated+cell+wall+biosynthetic+complexes+in+bacteria.">Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria.</a> Science. 333, 225-228 (2011).</li><li>Sparkes, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleach+it,+switch+it,+bounce+it,+pull+it:+using+lasers+to+reveal+plant+cell+dynamics.">Bleach it, switch it, bounce it, pull it: using lasers to reveal plant cell dynamics.</a> J. Exp. Bot. 62, 1-7 (2011).</li><li>Uchida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+synthesis+and+absolute+configuration+of+avenolide,+extracellular+factor+in+Streptomyces+avermitilis.">Total synthesis and absolute configuration of avenolide, extracellular factor in Streptomyces avermitilis.</a> J. Antibiot. (Tokyo). , (2011).</li><li>Yu, H., Wedlich-Soldner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+actin+dynamics:+Generating+randomness+by+formin(g)+and+moving.">Cortical actin dynamics: Generating randomness by formin(g) and moving.</a> Bioarchitecture. 1, 165-168 (2011).</li><li>Yu, J. H., Crevenna, A. H., Bettenbuhl, M., Freisinger, T., Wedlich-Soldner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+actin+dynamics+driven+by+formins+and+myosin+V.">Cortical actin dynamics driven by formins and myosin V.</a> J. Cell. Sci. 124, 1533-1541 (2011).</li><li>Berchtold, D., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TORC2+plasma+membrane+localization+is+essential+for+cell+viability+and+restricted+to+a+distinct+domain.">TORC2 plasma membrane localization is essential for cell viability and restricted to a distinct domain.</a> Mol. Biol. Cell. 20, 1565-1575 (2009).</li><li>Vizcay-Barrena, G., Webb, S. E., Martin-Fernandez, M. L., Wilson, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+and+single-molecule+imaging+of+plant+fluorescent+proteins+using+total+internal+reflection+fluorescence+microscopy+(TIRFM).">Subcellular and single-molecule imaging of plant fluorescent proteins using total internal reflection fluorescence microscopy (TIRFM).</a> J. Exp. Bot. 62, 5419-5428 (2011).</li></ol>

没有利益冲突的声明。

TIRF显微镜图像周边蛋白的首选技术。渐逝场的低渗透深度，最大限度地减少了光的焦点，这导致了一个非常好的信噪比，并允许数据采集，高帧频，或非常弱表达的蛋白质成像。高对比度和高帧率相结合，使TIRF显微镜的成像时空外皮本地化的蛋白质动力学的完美的工具。有趣的是，厚厚的细胞壁周围的许多微生物不妨碍周边蛋白的TIRF成像。事实上，渐逝的实际发电量很可能发生在细胞壁和细胞...

<table border="1"><tbody><tr><td> 该工具的名称/试剂 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>轨道摇床</td><td>工具</td><td> UniEquip </td><td> UNITWIST的3-D摇杆振动筛</td></tr><tr><td>的TIRF显微镜</td><td>直到</td><td></td><td>自定义安装</td></tr><tr><td>玻璃容器</td><td>工具</td><td> vitlab </td><td> 340-232880353 </td></tr><tr><td>陶瓷染色架</td><td>工具</td><td>托马斯科学。 </td><td> 8542E40 </td></tr><tr><td>刀豆蛋白A </td><td>试剂</td><td>西格玛</td><td> L7647 </td></tr><tr><td>的盖玻片＃1（18×18毫米） </td><td>显微镜</td><td>门泽尔格拉泽</td><td> BB018018A1 </td></tr><tr><td>显微镜载玻片</td><td>显微镜</td><td>门泽尔Ğ激光</td><td> AA00000102E </td></tr><tr><td>浸油</td><td>显微镜</td><td>蔡司</td><td> immersol 518F </td></tr><tr><td>琼脂糖</td><td>试剂</td><td> Invitrogen公司</td><td> 16500-500 </td></tr><tr><td> FluoSpheres </td><td>试剂</td><td> Invitrogen公司</td><td> F8795 </td></tr></tbody></table>

visualization of cortex organization and dynamics in microorganisms, using total internal reflection fluorescence microscopy

TIRF显微镜已成为一个强大的成像技术，研究时空动态的荧光分子在体外和活细胞1。全内反射光的现象时有发生，当光线从高折射率介质传递到与低折射率介质在一个角度大于特征的临界角（即接近边界平行）。尽管所有的光被反射回在这种情况下，创建渐逝波传播横跨和沿边界，距离指数衰减，只有穿透样本地区是100-200纳米界面附近的2。除了 ​​提供优越的轴向分辨率，减少了重点荧光激发创建了一个非常高的信号噪声比和减少漂白2,3破坏性影响。作为一个广角技术的TIRF还允许更快的图像交流quisition比扫描基于共聚焦设置。 乍一看，低的TIRF穿透深度，似乎是不符合，这往往是厚厚的细胞壁所包围的细菌和真菌细胞成像。相反，我们发现，酵母和细菌细胞的细胞壁，实际上提高可用性的TIRF和增加4-8观察到的结构范围内。许多细胞过程，因此可以直接访问的TIRF小，单细胞微生物，它往往提供了强有力的遗传操作技术。这使我们能够执行在体内的生化实验，可以直接在活细胞中进行评估蛋白质相互作用和活动的动力学。 在这里，我们描述了酵母或枯草芽孢杆菌细胞获得高品质的TIRF图像所需的各个步骤。我们指出的各种问题，可以affeCT的TIRF可视化细胞中的荧光探针和几个应用实例说明过程。最后，我们展示的TIRF图像可进一步提高使用既定的图像恢复技术。

Watch this Scientific Journal Video about Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy at JoVE.com

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

全内反射荧光显微镜（TIRF）是一个功能强大的方法来观察结构在细胞表面的高对比度和时间分辨率。我们展示的TIRF如何可受聘在皮层细胞壁封闭的细菌和真菌细胞的蛋白质动力学研究。

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

visualization-cortex-organization-dynamics-microorganisms-using-total

Research

JoVE Journal

Bioengineering

11.4K 次观看.  Max Planck Institute of Biochemistry. 全内反射荧光显微镜（TIRF）是一个功能强大的方法来观察结构在细胞表面的高对比度和时间分辨率。我们展示的TIRF如何可受聘在皮层细胞壁封闭的细菌和真菌细胞的蛋白质动力学研究。

视频: Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

重组DNA和蛋白质复合物，用原子力显微镜的可视化

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

科研

生物工程

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM). Cells are cultivated on transparent slides and incubated with a fluorescent marker homogeneously distributed in their plasma membrane. Illumination occurs by a parallel laser beam under variable angles of total internal reflection (TIR) with different penetration depths of the evanescent electromagnetic field. Recording of fluorescence images upon irradiation at about 10 different angles permits to calculate cell-substrate distances with a precision of a few nanometers. Differences of adhesion between various cell lines, e.g. cancer cells and less malignant cells, are thus determined. In addition, possible changes of cell adhesion upon chemical or photodynamic treatment can be examined. In comparison with other methods of super-resolution microscopy light exposure is kept very small, and no damage of living cells is expected to occur.

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

Topology of cell adhesion on a substrate is measured with nanometre precision by variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).

Nanotopology细胞粘附在可变角度的全内反射荧光显微镜（VA-TIRFM）

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence ...

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

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

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

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用：细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

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

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

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

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count.

用数字全息显微镜 (DHM) 定量化低浓度微生物

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

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

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

使用一个荧光显微镜进行多种成像模式

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

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

枯草芽孢杆菌中细菌体和内膜的可视化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...