作者要感谢欧金尼奥维瓦斯，库尔特Heungens，埃里克·马腾斯，查尔斯·考尔斯，美糖，埃里克Stabb，托德Ciche本协议和工具的发展所作出的贡献。 KEM，江铃支持由美国国立卫生研究院（NIH）国家研究服务奖T32（AI55397“微生物在健康和疾病”）。江铃汽车得到了美国国家科学基金会（NSF）研究生研究奖学金。这项工作是支持由美国国家科学基金会（IOS-0920631，IOS-0950873）的资助。

1。建设一个荧光的细菌菌株，通过共轭<ol><li>成长的受体菌（共生体进行审查）和供体菌过夜。供体菌株中，通常会大肠杆菌 ，应该是能够通过共轭捐赠DNA应转化的质粒（ 表2）的，携带编码荧光蛋白的基因。根据质粒上，共轭辅助菌株也可能是必需的。如果是这样的话，该菌株应还​​可以生长过夜。供体菌和辅助应变应种植与抗生素选择维护的质粒。 </li><li>继代培养的捐赠者，助手，受体菌营养丰富的生长介质，缺乏文化到介质中的比例为1:100的抗生素。 </li><li>每个菌株在温度适宜成长的文化，直到他们达到数中期阶段的增长（OD 600〜0.6）。 致病杆菌和大肠杆菌大肠杆菌这一阶段的成长次达到约3至4小时后，传代培养。这可能会有所不同，这取决于在该菌株中，或在某些情况下，该质粒，用。 </li><li>在离心管中，离心2分钟17,900 XG（13,000 RPM在最microfuges），混合菌株。供体和受体，产生最好的结果的比例会有所不同，不同的组合，并且可以具有可以凭经验确定。 ，对于嗜收件人和E.大肠杆菌供体，我们用一个3:1或1:1的比例。如果一个辅助菌（ 如大肠杆菌 ），它应该被添加到相同的比例作为供体。 </li><li>滗析出上清液。 </li><li>细胞沉淀重新悬浮在30μl的新鲜培养基。 </li><li>现货悬挂到营养丰富的传媒板块没有任何抗生素，并允许当场干燥。 </li><li>孵育板，倒立，一夜之间在一个受体细菌和允许捐赠者和帮手，（如适用）的最佳温度。板培养至少18小时。 </li><li>刮掉的斑点和条纹的选择性抗生素板的单菌落。抗生素应选择对供体和含有质粒的收件人。一个或两个额外的隔离条纹可能是需要分离的纯培养物。 </li><li>确保产生的菌落收件人共生体，而不是供体菌株，通过筛选存在的特殊的表型，如菌落形态，或收件人特定基因的聚合酶链反应检测。筛选对存在的质粒的菌落，通过聚合酶链反应的公知的质粒序列和荧光。菌落下的正确的波长对应的荧光蛋白的荧光。 </li></ol> 2。生产的无菌线虫虫卵<ol><li>细菌共生的自然增长5毫升过夜。要开始一种文化，细菌接种到溶原性肉汤（LB）或其他立方米lture媒体直接从冷冻柜股票或条纹板。 </li><li>消息传播600微升的细菌培养到10毫米脂质琼脂（LA）的板29上 。八至十个板块将产生足够的线虫大多数物种。 </li><li>孵育在25℃在黑暗没有水分两天。 </li><li>添加5000感染性线虫，或其他阶段取决于正在使用的线虫，在500μl的媒体到细菌的草坪。板在25℃下孵育3天。 </li><li>检查您的板存在的成年线虫和鸡蛋。到显微镜玻片上，将20微升的水。使用无菌棒刮少量的线虫从细菌的草坪。将棒入水，让线虫游泳。在低倍镜下看在您的幻灯片。对于外观的更多信息，请参阅讨论和图2。 </li><li>如果卵子和女性是可见的，继续，否则，孵化板25＆D例如，C和检查适当发展每6-12小时。当女性包含鸡蛋，放置几毫升水，在板的表面上。轻轻旋转板，将水倒入50毫升锥形管。线虫应该脱落的板，并不再是可见的板表面上。几个漂洗可能是必要的。 </li><li>允许线虫沉淀到该管的底部。 </li><li>冲洗线虫。移液器多余的水从该管的顶部。补充用干净的水和允许成人线虫解决。移液器多余的水。 </li><li>填写锥形管与鸡蛋的解决方案。一旦鸡蛋溶液中加入的所有步骤的定时用鸡蛋溶液必须继续进行与所述的相同的最大产蛋率。孵育管，同时轻轻摇晃了整整10分钟。大多数线虫应溶解的培养结束。 </li><li>立时离心机的圆锥形管正好是10分钟（这包括使离心机，以加速在1250 XG但没有下降到0×克。使用制动）。 </li><li>立即倾倒出上清。 </li><li>立即重新悬浮的颗粒用鸡蛋液，用移液管。 </li><li>立即填写锥形管中与鸡蛋的解决方案和拌匀反转的3-5倍。然后立即旋转在2.10。 </li><li>立即倾倒出上清。 </li><li>重新悬浮颗粒在LB移液转移到一个15毫升锥形管，以提高制粒过程中的洗涤步骤。 LB是斯氏菌的标准。根据线虫和细菌，同时要注意缓冲区必须不造成伤害的线虫或细 ​​菌，可用于其他缓冲区（ 例如，一个最小的盐介质）。 </li><li>填充管LB和自旋在2.10。 </li><li>滗析出上清液，并重新悬浮在LB。 </li><li>冲洗线虫共3次重复2.14和2.15。 </li><li>再悬浮线虫虫卵稀释至适当的体积。应该有至少10只蛋每微升。例如：gs的可以存储至少4天，通过在6厘米的陪替氏培养皿在5毫升LB培养基中加入抗生素，抑制定植细菌的生长，并在封口膜包装板。鸡蛋将需要洗涤一次，在15毫升的LB（如在2.13和2.14）的前，接种到细菌草坪。请注意，在此期间，卵孵化和使用时定植分析可能不同步发展。 </li></ol> 3。共培养法与荧光杆菌<ol><li>一夜之间荧光细菌成长，选择的质粒如有必要，。 </li><li>消息传播600微升的细菌培养到10毫米脂质琼脂平板上，用无菌的棒。在25℃孵育2天。 </li><li>发布500-5000无菌线虫鸡蛋在总共100至400微升（最佳2000的线虫在200μl）到脂质琼脂平板上每个。 </li><li>不含水分在黑暗中在25℃下孵育直至IJS，或其他感兴趣的，形式阶段。 IJS将是可见的为富中资源的白色环的板的边缘上。要在其他生命阶段，协议4。 </li><li>将板的变形的白色阱30。取下盖子的脂质琼脂平板上和放置到一个空的100毫米×20毫米陪替氏培养皿底部的底部。填写与水的大培养皿中，或其他适合您的线虫的缓冲，中小板包围。水位应该是约小板的高度的一半。 </li><li>孵育水到缓冲区中，直到后代的IJS已经出现的陷阱。 </li><li>在组织培养烧瓶中，水可被存储在感染性线虫幼虫。 </li></ol> 4。的生命早期筛查收集<ol><li>使用步骤3.1至3.4建立检测。 </li><li>孵育板，直到所需的生活阶段。每日目视检查，可能是必要的，以确定定时。要检查板，使用过程中描述的协议2.5。对于S. carpocapsae </eM&gt; eggsgrown 与 X 线虫上LA板块，怀孕的女性会在4-5天，未成年人将出现在7天，感染青少年，将开始生产的15-17天。 </li><li>当所需的生活阶段，采集样品。填补了的96孔板的孔中，用200μl的PBS中，或其他合适的缓冲液中。轻轻地刮去一些含有线虫的细菌草坪。豌豆大小的量（约50毫克），将提供至少几百线虫一旦产生后代，但更多的可能是必要的较早的时间点。将棒以及含缓冲。 </li><li>孵育30秒到一分钟。取出棒刮去剩余的残渣。使用棒，以消除任何琼脂从井里。线虫应该是在缓冲区内可见。将缓冲区混合物到一个干净的微量离心管中。 </li><li>治疗用左旋咪唑或其他瘫痪代理的线虫。几粒左旋咪唑溶解到30＆亩;升的水。每50μl的样品中加入1-2μ​​l这种混合。左旋咪唑治疗后，线虫将不可行。 </li><li>在以后的日子，如果样品将被视为解决所描述的这一步。如果没有，请跳到步骤4.6。加入200μl含有1X缓冲液和4％多聚甲醛固定液。轻轻混匀，移液可能会导致裂解精致生活阶段。用铝箔纸覆盖，，孵化的低振动筛在室温下至少18小时。 </li><li>将反应管的管架中，并允许线虫沉淀到该管的底部。 </li><li>冲洗线虫来去除背景。用移液管把多余的液体，添加200 - 500μl的缓冲，轻轻混匀。重复以上步骤。这可以重复几次，以去除更多的背景，如果需要的话。 </li><li>允许线虫沉淀到该管的底部，并重新悬浮在所需的量的。 </li><li>如果线虫是不固定的，屏幕上立即。可被存储在冰箱中最多固定线虫两个星期。存放在黑暗中。 </li></ol> 5。细菌在显微镜下观察协会的筛选线虫<ol><li>选择一些线虫在显微镜下查看你的混合物取出一个小样本。 </li><li>为了确保线虫的图片，为麻痹代理协议4.5中描述的治疗。如果你不拍摄照片或的线虫已固定，这一步可以省略。 </li><li> 20-30微升的线虫在水中添加您的显微镜幻灯片，并把顶部的盖玻片上。 </li><li>整个使用光学显微镜下的线虫，以确保线虫的视野。 </li><li>查看线虫使用荧光设置对应于由细菌表达的荧光显微镜。 </li><li>要确定细菌的本地化，在荧光灯下的设置和光镜设置，拍照的线虫，然后叠加图像。照片需要是相同的FIELD的观点。这可能需要尝试几种不同的放大倍率和意见的线虫的细菌。 </li><li>分布线虫定植可以量化通过计数线虫人口，得分为存在或不存在的细菌，并计算％定植。我们建议使用计数，直到至少30线虫内的人口在每个类别进行计数（带或不带定植），得到一个可靠的值。 </li><li>当只有少量线虫在人口定植，它可能是必要的30日前的观察计数几千线虫。对于高通量的计数，请使用以下步骤。 </li><li>相反单独计数的线虫，等分人口成多孔板（ 例如 24孔板）。后线虫已结算的菜的底部，各孔可扫描显微镜可以拿下和定植线虫的数目。 </li><li>线虫的总数我NA人口可以被识别的系列稀释线虫井。例如，1毫升的线虫可以被添加到一个井，充分混合搅拌用移液管尖，和3μl可以除去为定量的总人口在阱计数。 </li><li>定植由总数的线虫在阱的数目除以殖民线虫的百分比可以通过以下方式获得。 </li></ol> 6。代表性的成果 斯氏线虫与致病杆菌细菌相关的实施例的显微镜图像示于图3。要创建的合成图像， 如图3A所示 ，一个相位的对比度的图像的荧光图像重叠。 图3A中的箭头表示的细菌内的感染性线虫（巴= 100微米），以类似的方式构建， 如图3B示出了少年线虫的Wi日绿色荧光蛋白标记细菌（绿色棒）定位在整个线虫肠腔（巴= 20微米）。从两个媒体线虫的人口计数，取得了殖民细菌共生体（ 表1）。对于可靠的统计数据，最好是至少有30属于每个类别中，计算每个样品至少100线虫。在表1中可以看出，这些线虫定植上生长脂质琼脂和68.6％时，在约14.6％的水平，肝，肾琼脂上生长时。其他线虫和细菌物种已被证明有不同程度的殖民统治。例如X。线虫寄生99％的S. carpocapsae感染青少年（马滕斯2003年），P. 0813-124殖民26％的H.嗜菌感染的少年（Ciche 2003年）。 <img alt="图1" src="/files/ftp_upload/4298/4298fig1.jpg" /> 图1。纲要的方法。 A.细菌设计，以表达荧光蛋白。B.线虫虫卵从成人线虫分离生产无菌线虫C.无菌线虫与荧光细菌共培养。D.被视为下一个人生阶段的显微镜来评估范围内的线虫的细菌存在。 <img alt="图2" src="/files/ftp_upload/4298/4298fig2.jpg" /> 图2。含蛋的成年女性的描写。 A.该图显示了斯氏女性的整体外观。插图：DIC图像的S. feltiae妊娠的女性。黑色箭头表示外阴。白色箭头显示了明显的鸡蛋。图片是在放大20倍，比例尺为100μm。B. DIC图像的开发，但未孵化的S.feltiae线虫卵。图片的放大倍数是40倍，比例尺代表50μm。从S. C. DIC图像的鸡蛋分离feltiae 10倍的放大倍率下的线虫。比例尺为100微米。 <img alt="图3" src="/files/ftp_upload/4298/4298fig3.jpg" /> 图3。的显微镜图像线虫细菌的关联。 A. S. puntauvense线虫与细菌共生，X. bovienii，表达绿色荧光蛋白。该图像是从相同的视场重叠相位与荧光图像的对比度的图像所产生的合成图像。箭头表示荧光线虫主机内的共生菌。比例尺为100μm。B.图中显示的S. carpocapsae少年线虫与GFP表达X.线虫线虫肠道内的本地化。该图像是通过构造超过微分干涉对比度的图像的荧光图像叠加。图中标尺代表20微米。 <table border="1"><tbody><tr><td> 应变 </td><td> 线虫数与acteria </td><td> 总线虫计数 </td><td> 线虫的百分比olonized </td></tr><tr><td> S. puntauvense脂质琼脂</td><td> 30 </td><td> 205 </td><td> 14.60％ </td></tr><tr><td> S. puntauvense肝肾琼脂</td><td> 72 </td><td> 105 </td><td> 68.60％ </td></tr></tbody></table> 表1中。用于细菌的存在的线虫种群的实施例的得分，在该实验中，无菌S. puntauvense线虫生长，其表达GFP的共生体在不同的生长介质（血脂的琼脂，肝肾琼脂32）测试殖民缺陷。总共有至少一个hundreð每个样品数，并取得线虫细菌的存在。统计力量，应该算在每个类别至少有30线虫三个实验复制。 <img alt="表2" src="/files/ftp_upload/4298/4298table2.jpg" /> 表2中。荧光蛋白含有质粒。列表潜在的荧光蛋白插入质粒的细菌共生给出列出名称的质粒。包括的其它信息的荧光蛋白质编码，抗生素盒用于质粒保养，其他的使用说明，源的质粒。在括号中指出的浓度的浓度的抗生素用于X。线虫，每种质粒已成功地用于在任一的致病杆菌或，光 。更多的信息可以从著名的引文。根据被测试的细菌上，一些质粒可能无法正常工作，根据荧光蛋白，抗生素的选择，插入部位，或复制原点。上面列出的质粒包含不同的功能，可以使使用在感兴趣的细菌的。例如，小Tn7-KSGFP的插入到attTn7网站的染色体，而pECM20插入的X。线虫通过同源重组的染色体。或者，pPROBE质粒维持染色体外，和每个pPROBE质粒具有相同的骨干和荧光基团，但有不同的可选择的标记物或复制起点，以使它们能够使用在各种生物分类或突变体背景。

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没有利益冲突的声明。

这里所描述的协议提供了一种方法，一种线虫主机（ 图1）内的细菌的光学检测。此方法利用的光学透明度，线虫和荧光标记细菌的能力，使在体内分析的细菌内的线虫主机（ 图3）。具体而言，这种方法在其主机的标识细菌本地化的。横跨线虫种群的细菌定植的频率可以通过计数线虫种群和得分为细菌的存在，来确定（ 表1）。此方法是，可以用于研?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>脂质琼脂 （无菌） </td><td></td><td></td><td>营养肉汤8克，琼脂15克，5克酵母提取物，890毫升水，10毫升0.2克/毫升的MgCl 2。 6H 2 O，96毫升玉米糖浆溶液*，4毫升玉米油* 搅拌均匀介质，同时浇注板 *添加高压灭菌后的无菌成分</td></tr><tr><td>玉米糖浆溶液 （无菌） </td><td></td><td></td><td> 7毫升玉米糖浆，89毫升水 混合和高压灭菌器</td></tr><tr><td>鸡蛋的解决方案</td><td></td><td></td><td> 16.6毫升12％次氯酸钠，5毫升5M KOH，80毫升水</td></tr><tr><td>溶原性肉汤 （无菌） </td><td></td><td></td><td>酵母提取物5克，10克胰蛋白胨，5克盐，1升的水 混合和高压灭菌器</td></tr><tr><td>微型离心</td><td>费舍尔</td><td> 13-100-675 </td><td>将工作任何的离心持有的离心管</td></tr><tr><td>离心分离</td><td>贝克曼</td><td> 366802 </td><td>大台式离心机，持有15毫升和50毫升锥形管</td></tr><tr><td>无菌60毫米X 15毫米培养皿</td><td>费舍尔</td><td> 0875713 </td><td></td></tr><tr><td> 50 mL离心管</td><td>费舍尔</td><td> 05-539-6 </td><td></td></tr><tr><td> 15 mL离心管</td><td>费舍尔</td><td> 05-531-6 </td><td></td></tr><tr><td>无菌100毫米X 20毫米培养皿</td><td>费舍尔</td><td> 0875711Z </td><td>深于标准的培养皿</td></tr><tr><td> 24 - 孔板</td><td>格雷纳生物&lt;/ TD&gt; <td> 662000-06 </td><td></td></tr><tr><td>显微镜</td><td></td><td></td><td>荧光功能，兼容的荧光显微镜需要</td></tr><tr><td>多聚甲醛</td><td>电子显微镜科学</td><td> 15710 </td><td></td></tr><tr><td> PBS （无菌） </td><td></td><td></td><td> 8克氯化钠 0.2克KCl 1.44克Na 2 HPO 4， 0.24克KH 2 PO 4 1升的水 调整到pH为7.4，水至1 L和高压釜中</td></tr><tr><td>微型离心管</td><td>费舍尔</td><td> 05-408-138 </td><td> 2毫升或1.5毫升管</td></tr><tr><td>振动筛</td><td></td><td></td><td>任何振动筛，使液体轻轻移动工作</td></tr><tr><td>二氨基庚二酸</td><td>西格玛</td><td&gt; D-1377 </td><td>如果需要，补充介质的浓度或1mM </td></tr></tbody></table>

visualizing bacteria in nematodes using fluorescent microscopy

共生，生活在一起的两个或多个物种，广泛分布于所有的王国的生活。作为地球上的最普遍的生物，线虫和细菌形成共生广泛的范围从有利于致病1-3。一个这样的协会是互惠互利的关系致病杆菌的细菌和斯氏线虫，这已经成为了一个模型系统的共生4。 斯氏线虫，昆虫，细菌共生体，杀死昆虫5。昆虫宿主之间传输时，细菌定植于肠道线虫的感染青少年阶段6-8。最近，几个其他线虫种已被示出利用细菌杀死昆虫9-13，并调查已经开始研究在这些系统中的线虫和细菌之间的相互作用&lt;SUP&gt; 9。 我们描述了一种内共生菌或一种线虫主机上进行可视化的方法，利用通过显微镜观察时，光学透明度的线虫。这种细菌被设计来表达荧光蛋白，让他们通过荧光显微镜的可视化。许多质粒是可携带荧光在不同的波长（ 例如，绿色或红色），和共轭从一种供体到受体细菌共生体的大肠杆菌菌株的质粒编码的蛋白质的基因是成功的范围广泛的细菌。描述的方法进行调查之间的关联斯氏carpocapsae和嗜线虫致病杆菌 14。类似的方法已被用来调查其他线虫细菌协会9，15-18，因此，该方法是普遍适用的。 的METHOD允许的细菌内线虫的存在和定位的特性，在不同的发展阶段，深入了解协会的性质和过程的殖民统治14，16，19。微观分析揭示了两个殖民频率范围内的人口和本地化的细菌宿主组织，14，16，19-21。监测细菌内线虫的人群，如超声22或研磨23，可提供平均水平的殖民与其他方法相比，这是一个优势，但没有可能，例如，歧视的人群高频率的低共生体负载从人口低频率高的共生体负荷。判别的频率和负载的定殖菌筛选时，可能是特别重要或描述细菌殖民表型的突变体21，24。事实上，荧光显微镜被用于高通量筛选的细菌定植17，18中的缺陷的突变体，并且是不太费力的比其他的方法，包括超声处理22 25日至27日，和个别线虫夹层28，29。

Watch this Scientific Journal Video about Visualizing Bacteria in Nematodes using Fluorescent Microscopy at JoVE.com

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

要研究之间的互利共生<em&gt;嗜</em细菌和<em&gt;斯氏线虫</em监测细菌的存在和位置内线虫线虫的方法被开发。实验的方法，它可以应用于其它系统，带来工程菌表达绿色荧光蛋白和可视化，使用荧光显微镜细菌内的透明的线虫。

使用荧光显微镜可视化中的细菌线虫

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

visualizing-bacteria-in-nematodes-using-fluorescent-microscopy

Research

JoVE Journal

Biology

18.7K 次观看.  University of Wisconsin-Madison. 要研究之间的互利共生嗜

视频: Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

科研

生物学

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 ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

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


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

可视化的单分子荧光显微镜下的DNA复制

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

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

在Cytokineticž圈的活细菌超分辨率成像使用快速三维结构照明显微镜（F3D-SIM卡）

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Visualizing Stromule Frequency with Fluorescence Microscopy

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

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

可视化Stromule频率与荧光显微镜

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

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

分离绿色荧光蛋白系统可视化细菌在感染过程中传递的效应

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

使用前 Vivo 共聚焦显微镜可视化淋巴节点结构和细胞定位

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.