我们感谢的Alto实验室的有益讨论。我们也感谢德州大学西南医学中心，特别是克里斯·吉尔平博士，汤姆雅努谢夫斯基，和劳里·穆勒的分子和细胞成像设备在专业知识和咨询。我们也感谢Xionan董批判性阅读的手稿。支持这项工作是由美国国立卫生研究院授予AI083359 NMA

1。哺乳动物细胞培养<ol><li>正常大鼠肾（NRK），永生化宫颈癌（HeLa细胞），或其他适当的哺乳动物细胞系进行培养，在37℃下，5％CO 2，贴住细胞菜肴（ 见表1的任何试剂或光刻的玻璃屏栅盖玻片装置，在该协议中使用）在DMEM +10％FBS和1％青/链霉素。应采取预防措施，以维持一个无菌的环境中工作时，与培养的哺乳动物细胞。 </li><li>细胞的汇合根据您的实验时间线（0-5小时与 1-2天注射后）应调整至50％〜25％，分别为。 </li></ol> 2。显微注射<ol><li>准备所需的蛋白质，DNA，或小分子中的缓冲液的选择，取决于其细胞培养的顺从，最终体积为〜50微升。惰性荧光染料，如得克萨斯州红色或级联蓝为了纪念显微注射细胞注射染料和荧光探针的选择应该有非重叠的激发和发射波长。您用于显微注射所需的利息的分子的浓度应根据经验来确定，和荧光示踪染料常用在0.5-1.0毫克/毫升。通常情况下，若丹明或另一种荧光团共价连接到您使用市售的试剂盒的兴趣的分子，但是，其它的方法可以根据所使用的检测灵敏度。有关详细信息，请参阅下面关键的考虑因素。 </li><li>通过0.22μm的离心过滤器，过滤器的注入剂（〜50微升）。离心过滤器是优选的超过真空和/或关联的死体积的注射器过滤。 </li><li>产生显微注射针，高硼硅玻璃吸液管被拉到燃烧的布朗 - 微普勒以下制造商的指引。另外，显微注射法可以从供应商处购买，包括Eppendorf公司，这使得femtotip显微注射针针。 </li><li>小心移液4μl的注入剂到不受干扰显微注射针的使用Eppendorf公司20 Femtotips。确保无气泡内拉显微注射针。微量注射针进入显微操纵器臂连接到一个倒置显微镜的Eppendorf FemptoJet Injectman装入。好好检讨显微注射过程中，读者可以直接到以前的可视化实验杂志第8条。 </li><li>将网格盖玻片和培养细胞的活细胞菜入菜持有人倒蔡司显微镜。查找光刻的网格内的细胞生长，并注意“网格”的字母数字标识符，这将被用来在所有随后的步骤。理想的情况下，细胞在盖玻片的中心作为定位在盖玻片的边缘，随后的步骤中，将限制样品转移。 </li><li>入到位手动下喷射臂在盖玻片上，并使用显微操作器的操纵杆和控制，低级的注射针，直到的前端轻轻触及的培养的细胞的表面上并按下&#39;限制超出这一点，以防止针的运动，从而打破了脆弱拉硼硅玻璃。细胞显微注射执行每Eppendorf公司FemtoJet InjectMan用户手册的所有其他方面。显微注射技术最近被用来了解个别分泌的毒性蛋白，细菌9。 </li><li> Microinject所选择的网格的每一个细胞内。随着在〜50％汇合的细胞生长，人们可以期望〜80个细胞每600μm2的网格（例如示于图3A）。因此，荧光和电子显微镜检查时，将有一个n〜30-40的实验组和对照细胞。 </li><li>将活细胞到哺乳动物细胞培养孵化器所需的菜时间作为支配的实验装置。或者，人们可以直接从微量注射继续进行时间推移显微镜分析在固定之前等待感兴趣的特定的亚细胞结构的外观。 </li></ol> 3。荧光显微镜<ol><li>如果需要的话，进行活细胞的显微注射细胞的荧光测量的时间推移，在这之后，更换介质用2.5％戊二醛在0.1M二甲胂酸缓冲液（pH7.4），并在室温下孵育10分钟。但是，如果不需要进行测量活细胞，弃去细胞生长培养基，并用5毫升3.7％甲醛在室温下放置10分钟在1xPBS固定细胞。丢弃的固定液和更换，用5毫升1xPBS，继续执行步骤3.2。注意：它是不添加戊二醛直到荧光显微镜是完成后的关键。 </li><li>活细胞培养皿放置在荧光显微镜下与光刻的格点的盖玻片（共焦不需要氏脚步），并收集5-10明场和荧光图像的方格内的细胞（ 图4A）文件的安排。识别那些细胞已用惰性气体喷射染料（ 图4B）的激发和发射波长的显微注射。重要的是要获得的图像在不同的放大倍数（例如，10倍和65倍）中，为了便于在后续步骤中，涉及电子显微镜显微注射细胞识别。 </li><li>利用共焦荧光显微镜，显微注射细胞内的网格确定在2.5以上步骤收集了Z-堆栈。高倍率（100x）的需要的，以便将被确定的亚细胞结构，在电子显微镜的荧光图像关联。 </li><li>盖玻片与市售盖玻片去除流体从活细胞菜肴的底部除去，并放置在处女细胞培养皿中含有5毫升细胞侧1xPBS。盖玻片，然后处理电子显微镜，详细说明如下。 </li></ol> 4。透射电子显微镜<ol><li>替换1xPBS用2毫升2.5％戊二醛在0.1M二甲胂酸缓冲液（pH7.4）的缓冲液，并在室温下孵育10分钟。潜伏期较长，而没有必要的，将有助于维护细胞结构。注：单独甲醛固色剂EM分析是不充分的。 </li><li>移除固定剂和染色细胞与另外2毫升1％四氧化锇的0.1M二甲砷酸盐缓冲液30分钟，然后由二甲砷酸缓冲液洗涤三次，最后替换DDH 2 O所有的缓冲</li><li>标准EM协议之后，在梯度（70％至100％）的乙醇系列样品脱水。准备EPON的树脂和地方盖玻片细胞的EPON树脂填充管的顶部。允许在60℃下进行24小时的聚合通过将光蚀刻网格盖玻片细胞端笔关于到EPON的树脂，景点，以及网格图案的细胞被转移到EPON树脂在聚合过程中（例如在图3B中所示）。 </li><li>删除盖玻片从EPON树脂快速浸入液氮和/或沸水。检查表面的树脂，在双目解剖范围内定位的网格权益（注：电网拍打转让的镜像）。使用无菌的剃刀刀片或手术刀，修剪的EPON块向下使得只有网格的利益是在EPON树脂块的顶点。 </li><li>使用超薄切片机，修剪块获得连续切片，在所希望的厚度（〜70 nm的厚度为常规TEM和〜250 nm的厚度用于X线体层的TEM）。相反，部分醋酸铀和柠檬酸铅的标准协议。 </li></ol> 5。相关光学和电子显微镜<ol><li>移居他处的细胞的兴趣在前面的步骤中获得的电子显微镜图像。已位于一旦显微细胞的兴趣，使用高分辨率的共聚焦荧光图像，以确定区域内的单元格的兴趣。这些图像在高分辨率TEM。使用地标，如小区外围和核包络线，以确定荧光叠的z的图像对应的EM切片。 </li><li>您所选择使用的成像程序（例如图像J，Adobe Photoshop等），降低不透明度到50％，覆盖到与它相关联的TEM图像的荧光图像。通过调整规模（记住约束比例）和旋转的共聚焦图像以匹配的TEM图像的图像对齐。在荧光图像的对比度，锐度等的精细调整，可能有助于在对准。注：我们发现，异染色质，核膜，细胞边缘的一个很好的标志性建筑，以帮助在取向的两个图像。 </li></ol> 6。关键的考虑因素<ol><li>为了极大地有助于定位注入的小分子，如罗丹明或荧光素的荧光标记可被共价连接使用市售的试剂盒。异位表达亚细胞定位荧光标记蛋白，以显微注射前获得了更多信息。因此，荧光团和惰性显微注射示踪染料的选择是很重要的。 </li><li>选择用于显微注射的细胞都秉承盖玻片的中心靠近，并避免选择一个网格接近边缘。这将出现概率最大的，所关注的细胞被转移到用于电子显微镜的EPON树脂。 </li><li>汇合细胞很重要，作为一个完全融合的板显微注射细胞识别在电子显微镜非常困难。相反，种子细胞，使得他们达到〜50％汇合的短期实验当天，或〜25％汇合天的较长（1-2天）实验。使用的位置和形状的细胞，以及“空的空间作为地标定位的电子显微镜图像。 </li><li>共焦光学片的厚度应被调谐到的实验的需要，因此，应凭经验确定10。 </li><li>甲醛和戊二醛固定剂应该是档次最高的。甲醛将细胞固定，但保留这两种蛋白的荧光特性和小的荧光分子（如罗丹明或FITC）。但是，它不会保留的结构，允许EM分辨率高，。戊二醛保护膜和亚细胞结构的高度为EM分析sis文件，但会显着阻碍荧光。因此，两个步骤的固定是两种荧光的关键和EM显微镜。甲醛固定步骤可以省略，如果进行活细胞的测量，作为最终的固定可以通过戊二醛处理执行。 </li></ol> 7。代表性的成果这个程序能提供的能力，提供一个直接进入真核细胞的细胞质分子的利益，并监控其定位和/或影响细胞和organeller建筑毫米纳米多分辨率。在图2中所示的实验装置示意图，和程序。这种技术依赖于细胞生长的激光蚀刻玻璃网格盖玻片（ 图3A），后共聚焦显微镜，是能够传输的EPON树脂的兴趣，以及格子图案的细胞显微注射（ 图3B）。 一个典型的的显微注射的CLEM程序产生荧光图像和电子显微镜，相关时，允许亚细胞定位和超微结构分析。光刻的玻璃屏栅盖玻片上生长的细胞的显微注射后得到明亮的场图像（ 图4A）与分子的利益。惰性跟踪染料掺入区分显微注射的细胞从的非显微注射细胞（ 图4B）。得到的共聚焦显微镜的z栈使用连接到感兴趣的（ 图4C）的显微注射小分子荧光团或其他标识符。将样品用戊二醛固定，和处理电子显微镜。得到的电子显微照片，并覆盖到其相应的细胞上（ 图4D）。窝藏荧光信号的地区，在更高的放大倍率，更详细的检查（ 图4E </STRONG&gt;）。 <img src="/files/ftp_upload/3650/3650fig1.jpg" alt="图1"/> 图1。时间轴的显微注射CLEM程序。根据在实验装置中，可以在同一天进行显微注射​​法和荧光测量。准备样品的电子显微镜是最耗时的，由于长期孵育步骤，并会持续2-3天。 TEM分析和相关的荧光信号与电子显微镜图像，可以在一天之内进行。 <img src="/files/ftp_upload/3650/3650fig2.jpg" alt="图2"/> 图2。一般插图的的显微注射CLEM程序。 第1步：找到激光蚀刻玻璃网格盖玻片上的细胞生长和microinject分子的利益。 步骤2：拍摄时，可同时捕捉亮场图像和激光共聚焦荧光显微注射细胞栈Z-。 第3步：准备盖玻片为EM分析，并嵌入在EPON树脂。使用转让的格子图案，修剪块，使得细胞的顶点。 第4步：切连续切片超薄切片机。 第5步：用EM图像，荧光图像的关联。 <img src="/files/ftp_upload/3650/3650fig3.jpg" alt="图3"/> 图3网格模式有利于细胞识别的利益。 面板一个描绘了显微注射〜50％汇合的细胞生长;注意电网标识符AK。比例尺为100微米。 面板B从盖玻片聚合EPON树脂块上，这将是剖串联后，被识别感兴趣的网格，在解剖显微镜显示转移的网格图案。 面板C示出了准备传送的栅格图案切片。请注意，所转移的网格图案是在背面上的树脂块;可扩展Ë酒吧是1200微米。 <img src="/files/ftp_upload/3650/3650fig4.jpg" alt="图4"/> 图4。代表从显微注射相关的光学和电子显微镜的结果。 图A显示了明场显微镜的激光蚀刻玻璃网格盖玻片上的NRK细胞生长的箭头指示两个CLEM面板中的CD，可视化的细胞。 图B显示的荧光梯级蓝的惰性示踪染料，在这种情况下，使用以确定细胞的显微注射。 面板C示出了明场和荧光叠加签名的罗丹明标记的小分子。这里的两个单元板A＆B，D组表示CLEM明场，荧光显微镜和电子显微镜;箭头表示荧光泪小点成像在更高的放大倍率在E组中的箭头表示。比例尺的条件是低点：面板A＆B，100微米，50微米C＆D，，E，100纳米。

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没有利益冲突的声明。

这里介绍的方法能够直接提供纯化的蛋白质，核酸，小分子真核细胞的细胞质中，通过荧光显微镜和电子显微镜的相关性，并能提供超高分辨率分析。使用这种方法是简单而强大的，可以在内部进行，与许多现有的核心设施和/或适当配备的电子显微镜实验室。该技术也适合于活细胞，允许用户监视的动态实时荧光标记的分子，从而可以使用的时间推移显微镜和相关的断层电子显微镜。 <p class="...

<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 </td></tr><tr><td>光刻盖玻片和活细胞板</td><td> MatTek </td><td> P35G-2-14-CGR​​D </td><td></td></tr><tr><td>德州红</td><td> Invitrogen公司</td><td> D3329 </td><td></td></tr><tr><td>级联蓝</td><td> Invitrogen公司</td><td> D7132 </td><td></td></tr><tr><td>罗丹明标记试剂盒</td><td> Thermo Scientific的</td><td> 53002 </td><td></td></tr><tr><td> 0.22微米离心过滤单元</td><td>密理博</td><td> UFC30GV00 </td><td></td></tr><tr><td>高硼硅玻璃吸管</td><td>萨特仪器</td><td> BF100-50-10 </td><td></td></tr><tr><td>微管牵引机</td><td>萨特仪器</td><td>型号：P-97 </td><td>在执行斜坡测试使用程序＃4 </td></tr><tr><td> FemtoJet显微注射系统</td><td> Eppendorf公司</td><td> Injectman NI2 </td><td></td></tr><tr><td>盖玻片切削液</td><td> MatTek </td><td> PDCF OS 30 </td><td></td></tr><tr><td> Technai透射电子显微镜</td><td> FEI </td><td> Technai G2 BioTWIN </td><td></td></tr><tr><td> Ultramicrotombe </td><td>徕卡</td><td> EM UC6 </td><td></td></tr></tbody></table>

correlative light and electron microscopy (clem) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets

依赖于复杂的真核细胞中，高稳定，功能不同的膜结合隔间保持必要的生化极性适当的细胞功能。因此，了解的酶，蛋白质和细胞骨架，治理和维护这种生化分离是非常重要的。使用荧光标记的分子定位和/或扰乱细胞内的车厢已经取得了丰富的知识和先进的细胞调控的认识。荧光和共聚焦显微镜成像技术，如确定一个荧光标记的小分子相对简单的位置，但是分辨率非常小的结构受限情况说明1。 另一方面，电子显微镜揭示了亚细胞形态的细节，以非常高的分辨率，但它的静态特性使得它难以衡量的高度动态的亲精度2,3 cesses用。因此，光显微镜和电子显微镜对同一样品，被称为的相关光学和电子显微镜（CLEM）4,5的组合，能提供超快荧光成像的双重优点，高分辨率的电子显微镜6。这种强大的技术已经实施，以研究细胞生物学的许多方面的5,7。公司自成立以来，这个过程增加了我们的辨别能力，在高分辨率下的亚细胞结构和形态。 在这里，我们提出了一种简化的方法进行快速显微注射，其次是CLEM（ 图1）。显微注射的CLEM程序可以使用，引入特定数量的小分子和/或蛋白直接进入真核细胞的细胞质中，研究了从毫米到纳米多分辨率（ 图2）。该技术是基于显微注射细胞生长的激光蚀刻玻璃网格底部的活细胞菜肴和成像，共聚焦荧光和电子显微镜的盖玻片贴。容易的格子图案，这是很容易转移，以及所关注的细胞，EPON用于固定样品的树脂切片前的电子显微镜分析（ 图3）的细胞的本地化（s）的权益。叠加的荧光灯和EM的图像允许用户确定的亚细胞定位，以及任何形态和/或超微结构的变化引起的显微注射分子的利益（ 图4）。此技术是适合从≤5 s到几个小时，取决于的显微注射样品的性质上的时间点。

Watch this Scientific Journal Video about Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets at JoVE.com

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

CLEM技术已被用于分析超微结构形态的细胞膜，细胞器，通过显微注射的分子和亚细胞结构的影响。这种方法结合了强大的技术，的显微/显微注射，共聚焦荧光显微镜，电子显微镜，允许毫米多纳米的分辨率。此技术是适合于各种各样的应用。


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

correlative-light-electron-microscopy-clem-as-tool-to-visualize

Research

JoVE Journal

Biology

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

19.0K 次观看.  University of Texas Southwestern Medical Center. CLEM技术已被用于分析超微结构形态的细胞膜，细胞器，通过显微注射的分子和亚细胞结构的影响。这种方法结合了强大的技术，的显微/显微注射，共聚焦荧光显微镜，电子显微镜，允许毫米多纳米的分辨率。此技术是适合于各种各样的应用。

视频: Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

作为寄主植物挥发物的筛选工具electroantennographic生物活性

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

科研

生物学

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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 ...

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell&#8217;s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.

Here, we introduce a method, cocem3D, to unveil the ultrastructure of a specific cell in its native tissue by bridging confocal and serial block-face scanning electron microscopy.

一个具体的感觉细胞的共聚焦相关和3D电子显微镜

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

扫描浸软组织的电子显微镜可视化的细胞外基质

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

用化学干燥进行扫描电镜 (sem) 形态分析的原核生物和真核生物的制备

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

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.

使用新型机器人系统 Spotiton 进行时间解决研究的低温电子微观网格准备

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view.
In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification.
In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.

Here, we present a protocol for optimized on-section correlative light-electron microscopy based on endogenous, fluorescent labeling as a tool to investigate the localization of rare proteins in relation to cellular ultrastructure. The power of this approach is demonstrated by ultrastructural localization of endogenous LC3 in starved cells without Bafilomycin treatment.

内源性LC3的超微结构定位-截面相关光电子显微镜

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal ...

同时保存荧光和超微结构的新方案

Epon Post Embedding Correlative Light and Electron Microscopy

Correlative light and electron microscopy (CLEM) is a comprehensive microscopy that combines the localization information provided by fluorescence microscopy (FM) and the context of cellular ultrastructure acquired by electron microscopy (EM). CLEM is a trade-off between fluorescence and ultrastructure, and usually, ultrastructure compromises fluorescence. Compared with other hydrophilic embedding resins, such as glycidyl methacrylate, HM20, or K4M, Epon is superior in ultrastructure preservation and sectioning properties. Previously, we had demonstrated that mEosEM can survive osmium tetroxide fixation and Epon embedding. Using mEosEM, we achieved, for the first time, Epon post embedding CLEM, which maintains the fluorescence and the ultrastructure simultaneously. Here, we provide step-by-step details about the EM sample preparation, the FM imaging, the EM imaging, and the image alignment. We also improve the procedures for identifying the same cell imaged by FM imaging during the EM imaging and detail the registration between the FM and EM images. We believe one can easily achieve Epon post embedding correlative light and electron microscopy following this new protocol in traditional EM facilities.

作者聚焦：相关光学和电子显微镜与荧光蛋白保存的进步

We present a detailed protocol for Epon post-embedding correlative light and electron microscopy using a fluorescent protein called mScarlet. This method can maintain the fluorescence and the ultrastructure simultaneously. This technique is amenable to a wide variety of biological applications.

相关的光学和电子显微镜（CLEM），作为工具来可视化显微注射分子和他们的真核细胞的亚细胞目标