该项目号634429.本刊物只反映作者本人的观点赠款协议下收到的资金来自欧盟的地平线2020年研究和创新计划，以及欧洲委员会不能承担责任可能作出的任何信息使用所载。我们也承认在真正的哈尔丁植物园，中船重工做出的财政贡献。 SR感谢欧盟[ITN-SAPRO-238550]为支持她的水霉的研究。我们还要感谢旧金山德卡隆赫友好地处理样品（图5）提供Phellorinia herculanea图像和B.普埃约。所有图像都采取了在Real哈尔丁植物园 - 中国船舶重工集团公司在马德里的SEM服务。

注:本协议包括六个主要部分，三专门为特定的生物（第1-3款），以及三个描述程序都（4-6）常见。星号（*）表示实验者修改的步骤。 1.制定研究和完全形成植物结构<ol><li> 收集和固定 <ol><li>如果该植物材料被收集在一个地方，没有访问一个通风橱，介绍并浸泡在70％乙醇中的材料离心管中。理想的情况下，浸入该材料在美国联邦航空局（步骤1.1.1-1.1.3）48小时后，以避免在乙醇过度脱水。如果通风橱是植物材料可以访问，请忽略此步骤并继续1.1.1。 </li><li>在装有醛滤波器通风橱制备福尔马林乙醇（FAA）固定剂。添加85份的70％的变性乙醇，10份的60％的甲醛溶液，以及5份冰醋酸的。准备FAA刚刚杀青的材料，作为其长期储存之前，不建议40。 </li><li>在通风橱，倒FAA的股票进入各个广口防漏塑料瓶。因为有提供样品使用尽可能多的瓶子，并创建样品标识标签。 </li><li>选择花卉或营养分生组织修复，从而确保它们不会被昆虫，真菌，或极端的天气条件损坏。切枝，除去不需要的材料，并立即沉积在样品中的FAA的溶液。 </li><li> 72-96小时后，倒入FAA转化为化学处理的塑料容器。立即，洗样品三次以新鲜的70％乙醇，以除去任何残留的FAA的。固定材料可以无限期储存在70％的乙醇。 </li></ol></li><li> 解剖和脱水 <ol><li>使用超细镊子解剖在70％乙醇固定材料的体视显微镜下，NEedles，镊子，刷子，和微型手术刀（组织的最大尺寸应约1 cm 3时，或2厘米扁平材料）。解剖样本放进有盖的乙醇，以防止组织从干燥培养皿。使用培养皿覆盖有干燥的黑硅基地，以更好地看到反差的白色组织。 </li><li>放入样品容器中的解剖材料的临界点干燥器（CPD， 图2a）。此时，浸入容器放入培养皿，用70％的乙醇，并且包括样品标识标签（用纸和笔制造）。为了更有效的干燥用于进一步操作，避免在同一容器中混合的年轻和成熟的样品。* </li><li>放置在容器的盖和用大量的70％乙醇将它们存放在塑料离心管中。过夜储存管，如果不立即处理的材料。 </li><li>通过以下乙醇甲级传送解剖材料S IN密封罐或离心管:70％，90％，100％和100％。离开样品中至少1小时各溶液。保持样品过夜在100％乙醇溶液中。 </li><li>与材料的CPD（第4节）转移容器。 </li></ol></li><li> 安装和准备的植物组织的SEM观察 <ol><li>写SEM样品架（即铝存根）下的样品标识号。覆盖双面胶带存根的顶部。放置存根成试样保持器（ 图2b）。 </li><li>在立体显微镜，小心地打开携带的CPD已经干燥娇嫩的样品容器。请记住，在CPD处理后，样品变得更轻和敏感的静电。关闭一旦样本已经取出，以避免灰尘或杂质的容器。 </li><li>把样品存根的粘物表面上，规划未来所需位置（一旦样品触摸表面，这是非常难以去除它们）。不要试图携带在这一点上大清扫;只是删除多余的组织，很容易回升。对于孢粉研究，解剖花药和打开它们暴露在存根花粉。 </li><li>把长的样品（ 例如，长2厘米），诸如在水平位置花序。如果可能的话，同样的结构极，侧面和底部的意见东方样本。在存根留样本之间足够的空间。 </li><li>如果样本不能立即处理，让他们在一个密闭容器保护一夜之间用硅胶，以避免补液（ 图2c）*。涂层用溅射镀膜机，并将它们传送到SEM的样品（第5和6）。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/55031/55031fig2.jpg" /> 图2:工具样品manipulati并SEM观察前处理。 （ 一 ）钢铁制造与有孔的墙壁在CPD室中的乙醇/ CO 2交换标本容器。 （b）一种塑料试样保持器内钢存根。 （c）使用玻璃容器保持在湿度和防尘的样本。在碱，为硅胶的隔室。 （ 四 ）临界点干燥机。在前面，有（左到右）的压力计，电源开关，温度控制系统，和温度显示。对CO 2 -乙醇互换平时工作压力为60巴（800磅）。在顶部，有四个阀（入口，漏，通风和排气的控制）侧翼的中心样品室中。照片是采取Y.鲁伊斯 - 莱昂和MA贝洛。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55031/55031fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> &lt;<html> p系列="jove_title"&gt; 2。研究不同的表面水霉 （卵菌纲）囊肿行为<ol><li> 成长和固定囊肿 <ol><li>制备蛋白胨和葡萄糖（PG-1）用D-媒体41 - （+） -葡萄糖（6克）和真菌蛋白胨（3克）*。加起来900毫升自来水和高压釜中于121℃40分钟。倾50毫升预先高压灭菌溶液A（的NaH 2 PO 4，0.13 M）和50毫升的溶液B（ 的 Na 2 HPO 4，0.13 M）。 </li><li>从股市文化上保持蛋白胨，葡萄糖琼脂培养基（PGA，这是准备为PG-L，但加入10代欧洲细菌琼脂的葡萄糖和釜前的蛋白胨） 水霉寄生株，生长菌丝菌落0.5毫升的PG-L的液滴在20℃下在培养皿中24-48小时。通过用高压灭菌自来水洗涤菌丝体三次，并在20℃温育它们15小时Ç诱导孢子形成="外部参照"&gt; 42，43。 </li><li>轻轻吹打悬浮液的上部收集所释放的副游动孢子并集中他们在1mL部分。涡旋产生二次囊肿44大力摇动30秒的游动孢子。 </li><li>为了测试孢囊的棘差动生长，在单独的陪替氏培养皿（P60），把0.5毫升二次囊肿悬浮液到不同的表面（ 即，碳，金，和铜TEM格栅上;鲑鱼和鳕鱼鳞（先前漂白）和玻璃盖玻片）*。孵育孢囊在20℃下70分钟，这有利于孢囊至表面的附着。 </li><li>除去液体，并添加0.5毫升的2％戊二醛的每个表面的囊肿的固定。保持在室温下将样品在通风橱下1小时。 </li><li>除去戊二醛和通过乙醇系列（30％，40％，50％，60％，70％，80％脱水样品，90％，100％，100％），加入15分钟每乙醇溶液中加入5毫升。一旦在过去的100％乙醇溶液中，样品可以储存高达在密封的培养皿一个月。在这个阶段，将样品准备在CPD被干燥。 </li><li>小心地从培养皿转移网格和鳞为适合于CPD（第4部分）的支架。对于此步骤，利用一个CPD格架或堆叠试样保持器，它保持彼此分离的样品。就拿电网和尺度用镊子，牢记囊肿应朝上的网格所有的时间。* </li></ol></li><li> 安装和准备囊肿样品SEM观察 <ol><li>安装在先前覆盖的双面碳胶带和下方标有铝存根的网格和鳞。 </li><li>转移样品至溅射镀膜机（部分5）。 </li><li>观察下的SEM（第6）样本。 </li></ol></li></ol>Phellorinia herculanea的植物标本馆真菌孢子<ol><li> 再水化和孢子脱水 <ol><li>认真总结每个样品用滤纸，形成铅笔标记的信封〜0.5-1厘米2，注意不要粉碎他们。密封采用回形针滤纸。转移填充样品至培养皿并沉浸其中在10毫升水再水合周围孢子组织。 </li><li>立即把样品在微波炉（600W约20秒）。除去材料一旦水开始蒸发，并允许它在室温下冷却。 </li><li>通过以下乙醇系列传递样本:30％，50％，70％，80％，90％，95％，100％和100％。取决于样品的量，使用烧杯或离心管用于此步骤。离开样品在每种溶液15分钟。 </li><li>放置样品的CPD（第4节）。 </li></ol></li><li> 安装和p练习器孢子SEM观察<ol><li>打开信封。倒上用双面胶带预先制备的存根的孢子。另外，收集与存根的表面粘孢子，注意不要粉碎他们*。 </li><li>如果样品含有少量的孢子，除了先前的工序中，切成小片的信封（约1 平方毫米），并且将其放置在一个新的存根*。 </li><li>放置组织进入溅射镀膜机（部分5）。 </li><li>根据扫描电镜（第6）观察。 </li></ol></li></ol> 4.干燥材料使用临界点干燥器（CPD，图2d） <ol><li>使用CPD在通风良好的地方，并验证机的所有阀门都关闭。检查样品室是空的，干净的。 </li><li>开动机器，并确认温度控制系统测试自动发生。如果CPD有一个外部制冷浴系统，交换我前检查水位T ON。 </li><li>遵循用于乙醇和CO 2交换特定的CPD的制造商的说明。为了安全起见，进行训练的人在使用机器的监督下这一步。请记住，它暴露于快速的压力变化，这可能猛烈吹熄。 </li><li>取出样品，并继续执行步骤1.3如果与植物组织工作，步骤2.2如果与卵菌囊肿工作，步骤3.2如果与真菌孢子的工作。 </li></ol> 5.金使用溅射镀膜机（图3a）涂层的样品<ol><li>检查溅射镀膜机。验证金阴极靶处于良好状态。使用90％的乙醇普照的无绒布如果需要清洁真空室的墙壁和室盖。 </li><li>标记与用于显微镜下的样品的进一步鉴定每个末梢孔旁的数字溅射持有者。小心，放置装有SAMPL存根ES和固定。使用旋转的行星样品台，以确保与不规则表面标本均匀的涂层。 </li><li>按照制造商的说明来调整设置，如工作距离，操作气体压力（ 例如，5×10 -1 - 7×10 -1毫巴）（ 例如 ，30mm以下。），溅射时间（ 例如，50秒），金层（例如 12纳米）的当前（ 如 ，15个mA）的和电压源（ 例如，600伏）45的厚度。 </li><li>删除存根，并带他们到SEM（第6节）。可替代地，将存根与硅胶（ 图2c）的密封容器中。 </li></ol><img alt="图3" src="/files/ftp_upload/55031/55031fig3.jpg" /> 图3:溅射镀膜机（a）和扫描电子显微镜（b）中。 （a）该真空腔（左）的前视图，气体VALV即，定时器，真空，和电流控制。 （ 二 ）用SEM主要部件的侧视图（从左至右）:真空柱与样品室中，在计算机屏幕的控制，并且该腔室的监视器。照片是采取Y.鲁伊斯 - 莱昂。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55031/55031fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 6.扫描下观察电子显微镜（SEM，图3b） <ol><li> SEM启动 <ol><li>遵循制造商的指示来启动，并设置扫描电镜，调整样品的高度的目标孔径直径（ 例如，对于植物2微米，真菌和卵菌4微米），工作电压（ 例如，15千伏）。 </li><li>检查电子束系统的正确对准和设置的轴向对准并根据生产商的指示的stigmators。调整Ť他的工作，以获得场的适当深度的距离。 </li></ol></li><li> 图像采集 <ol><li>得到样品的聚焦图像，并使用它作为起点。增加放大率接近最大水平并再次聚焦影像。选择与表面凹凸的领域，如孔。正确散光并调整最佳对比度和亮度。 </li><li>捕获的SEM图像的高分辨率。使用BSE检测如果图像显示，样本充电。否则，设置SE探测器。改变按照制造商的指示检测器。 </li></ol></li></ol>

The authors have nothing to disclose.

相对于标准的SEM协议，这里提出的步骤包括相对快速的，容易执行，和低成本的方法。取决于样品的量和在便于加工，需要四到五天以获得良好质量的图像。包括为CPD和SEM的操作足够的安全预防措施，程序很容易处理。尤其应谨慎用福尔马林和戊二醛采取（参见1.1.1步骤1.1.3来和协议的2.1.5）。有一定的步骤，其中，如果需要的话，该过程可以停止一段长的时间，而不会损坏样品或破坏前面的步骤（ 例如，步骤1.1.5，1.2.3和1.3.5）。在成本方面，许多设备的可重复使用几制剂（ 例如，CPD铝容器和存根持有人），和所用试剂可从所有的商业供应商的非昂贵的化学品。 这些程序的缺点是需要适当的化学废物处置的醛和乙醇以及缺乏关键用品和技术的限制。材料如金磁盘的溅射镀膜机和CO 2筒的CPD应提前好进行检查。如果他们是在连续使用，实验室将需要他们的多支股票，最终提升了成本。因为个别的研究人员往往不能证明从维护这些类型用品的衍生的费用，或者它们根本不需要连续使用扫描电子显微镜，这些程序时下趋向于与外部电子显微镜服务实验室中进行。 SEM的观察技术仅限于表面的高倍率研究。如果内部组织需要被观察到的，样品应在探索内部组织的外观的正确方法进行切割。为了探讨在高毫安电池内部方面gnification，透射电子显微镜（TEM）是必需的。在协议中的关键步骤是CPD治疗，它是保持组织免受令人震惊的变化，并与空气直接接触的关键前样品的适当固定和脱水。另外，在CPD样品室中的压力变化和涂层的沉积在不同类型的样品的量的细致的管理是很重要的。 尽管有这些限制和特殊操作，用SEM下列这里描述的方案的生物样品的研究允许对一些常见的问题，如细胞壁和器官失真（ 图1，图4，图5），对观察的限制分辨率和结构的生长从湿和液体环境来（ 图5,6），以及敏感的细胞（ 图1C，1D）的破坏。 这里介绍的协议已经allowe的D传统分类的特点，如在水霉 47囊肿脊柱结构重新解释和质疑。不同的表面上的水霉刺的差分增长模式说明了该功能的不稳定性及其对种诊断的局限性。除了用于分类学研究，器官发育阶段，感染性疾病，之前已探索由于这些技术从未观察到组织的功能相关特性的捕获。现在，该协议可以扩展到要检查33成千上万等待案例研究。据同行评审文献数据库SCOPUS，在最近过去的五年里已经出现了处理厂的SEM图像（4914）7,425篇出版物，卵菌（21）和真菌（2,490）。这一事实表明，研究使用SEM成像卵菌还是很稀缺与比较植物和真菌。用ESEM，数量也更低。有588手稿在相同的时间跨度:337在植物，1在卵菌和250中的真菌。

在生物学上，利用扫描电子显微镜（SEM）已经扩展到构造演化，比较形态学，器官发育，以及人口或物种1表征的研究。随着微观结构的二维视图，如微观和系统学区从SEM技术的进步，因为在20 世纪下半叶获利。例如，引入在20世纪70年代的溅射镀膜方法制成精巧的材料可能观测诸如茎尖和花增强非导电组织2,3的摄像。 SEM使用从试样的表面喷出重现地形在高真空环境中4个电子。 在涉及SEM研究主要集中在结构特征既推理和growt重建^ h的进程。而相关的分类结构的新字符的范围广泛的生物系统学已经从SEM的观察发现。例如，用于物种诊断或supraspecific分类植物性状，如木材5，柱头多样性6，蜜腺和花形态7,8，毛状体细节9和花粉的笼罩下的凹坑10，11，将不能正确而不可视SEM。与传统的SEM观察的成功已经也实现了长期福尔马林固定的生物体12和植物腊叶标本13。 在另一方面，利用扫描电镜生长过程重构研究涉及广泛的议题，如器官发育14的INFE由细菌15，植物根系生理16，寄生虫主机连接机构17，18日 ，寄生虫19，重寄生和抗菌20，21，生长畸形22，野生型和突变的个体23的比较发展和整个生命周期的药物作用引起的ctions 24。虽然环境扫描电子显微镜（ESEM）25可以具有重要的优点为湿的生物样品在生长过程中的观察，细腻的材料仍然可以甚至在ESEM）的低真空条件损害，并且需要适当地处理，以避免损失宝贵的形态学观察。 在本文中，具体的协议三种差异的SEM观察审查erent类型的样品，提出:花分生组织，卵菌（ 水霉 ）和真菌的材料。这些协议编译我们以前基于SEM研究26，27，28，29，30，31，32，33，其中的具体困难和替代解决方案已经发现的经验。在植物发育的比较和结构研究的情况下，利用SEM起步于上世纪70年代34，35，从那时起，研究人员发现，某些花的特点是比以前认为的36更不稳定。花发育的改造涉及到年轻的花分生组织和花之间的所有阶段的捕获。为了达到这个目的，它是ESSE微分方程边值问题的样品形貌和细胞壁完整性固定和随后的脱水后，不会受到损害。年轻的花分生组织特别容易被细胞壁塌陷（ 图1a，1b）。同样，结构精细，如蜜腺，花瓣，柱头和孢子囊需要有效和undamaging协议。本文总结了一个最佳的方案，以保持娇嫩的组织完好SEM成像。 在卵菌（原生藻菌）寄生虫最多样化的和广泛的基团的酮，与主机从微生物和植物，以无脊椎动物和脊椎动物37的情况下-有一些生长和在潮湿环境中培养孢子。这一条件表示为SEM观察的挑战，因为孢子需要适当的衬底不适合标准的SEM协议。间的卵菌纲， 水霉的种是特别感兴趣的，因为它们的Cañ引起aquacultures，渔业和两栖动物数量严重38减少。微形态特征，如包囊的钩状刺，已发现是确定水霉，这是基本的，以建立感染的控制和潜在的治疗39的种类是有用的。这里，有一个实验方案来比较包囊在不同基材上的脊柱生长的图案和操纵为临界点干燥器（CPD）制备和随后的SEM观察样品。 在第三情况下，存在的真菌Phellorinia herculanea f的孢子的检查之后想出有趣的发现。 斯泰拉塔 F。新星（伞菌）31。连同孢子，一组苗圃意外的细胞是在SEM下确定的。与以往传统的协议和未经处理的材料，护士进来细胞欧ŧ完全坍塌（ 图1c）。关于相关的孢子特定组织进一步推论可以用简单但重要的修改，以这里所描述（ 图1d）的标准方法进行。 在该评价中，有可以使用的处理与在SEM观察相关联的不同问题的详细SEM协议被子植物，卵菌和伞菌，如细胞瓦解和分生组织萎缩，囊肿棘非最优生长，破坏短暂的组织，分别为。 <img alt="图1" src="/files/ftp_upload/55031/55031fig1.jpg" /> 图1:无（A，C）和（B，D）的协议，FAA -乙醇- CPD处理样品的比较。 （A - B）Anacyclus棒曲霉，中期发展的花蕾。芽四氧化锇46 &lt;处理/ SUP&gt;（ 一 ）和巴德与FAA-CPD协议（ 二 ）处理。 （C - D）护士细胞Phellorinia herculanea的F孢子。 斯泰拉塔。干燥的样品未经任何处理（c）和与伞菌（ 四 ）这里所描述的协议。孢子为橙色。秤:（AB）100微米，（CD）为50μm。照片是采取Y.鲁伊斯 - 莱昂。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55031/55031fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table border="0" cellpadding="0" cellspacing="0" width="870">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Acetic acid</td>
			<td style="width:196px;">No specific supplier</td>
			<td style="width:131px;"></td>
			<td style="width:208px;">Skin irritation, eye irritation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">aluminium stubs</td>
			<td>Ted Pella, Inc.</td>
			<td>16221</td>
			<td>www.tedpella.com</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge tubes</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Critical Point Dryer</td>
			<td>Polaron Quatum Technologies</td>
			<td>CPD7501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D (+) Glucose</td>
			<td>Merck</td>
			<td>1,083,421,000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double sided sellotape</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol absolute</td>
			<td>No specific supplier.</td>
			<td></td>
			<td>Flammable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">European bacteriological agar</td>
			<td>Conda</td>
			<td>1800.00</td>
			<td>www.condalab.com</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter paper</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formalin 4%</td>
			<td>No specific supplier.</td>
			<td></td>
			<td>Harmful, acute toxicity, skin sensitisation, carcinogenicity. Flammable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass cover slips</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass hermetic container&#160;</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutaraldehyde 25% DC 253857.1611&#160; (L)</td>
			<td>Dismadel S.L.</td>
			<td>3336</td>
			<td>www.dismadel.com</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mycological peptone</td>
			<td>Conda</td>
			<td>1922.00</td>
			<td>www.condalab.com</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">needles</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic containers</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sample holder with lid&#160; for the critical point dryer&#160;</td>
			<td>Ted Pella, Inc.</td>
			<td>4591</td>
			<td>www.tedpella.com</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scalpels</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scanning Electron Microscope</td>
			<td>Hitachi</td>
			<td>S3000N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software for SEM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Solution A: NaH2PO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Solution B: Na2HPO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Specimen holders</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sputter coater</td>
			<td>Balzers</td>
			<td>SCD 004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomicroscope</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transmission Electron Microscope (TEM) grids</td>
			<td>Electron Microscopy Sciences</td>
			<td>G200 (Square Mesh)</td>
			<td>www.emsdiassum.com</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezers</td>
			<td>No specific supplier</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

scanning electron microscopy (sem) protocols for problematic plant, oomycete, and fungal samples

在生物样品的用于与扫描型电子显微镜（SEM）的观察处理常见的问题包括细胞崩溃，治疗从湿微环境和细胞破坏样品。用年轻的花组织，卵菌囊肿，真菌孢子（伞菌）为例，具体的协议来处理这里描述了克服一些在样品处理的SEM下图像采集的主要挑战细腻的样本。 固定FAA花分生组织（福尔马林乙醇），并与临界点干燥机（CPD）进行处理并没有显示倒塌细胞壁内或扭曲器官。这些结果对于花发育的重建的关键。从湿微环境，如戊二醛固定的卵菌囊肿样品的类似的基于CPD处理，是最佳的，以测试的诊断特征在不同类型的su差动生长（ 例如，囊肿刺）bstrates。补液，脱水和CPD处理，这些细胞的进一步的功能研究的一个重要步骤后避免附着到真菌孢子护士细胞的破坏。 这里详细的协议代表了低成本和收购的良好品质的图像重建生长过程和研究诊断的特点迅速的替代品。

花发育和发展的固定和完全形成植物结构使用这里描述的FAA-CPD协议，年轻而成熟的植物组织中得到最佳的固定和脱水SEM成像。过程如花发育可以重建，因为芽的地形和形状不被细胞萎缩（ 图1b，1d中，图4a-f）的失真。具有复杂形状的结构可以成功地覆盖有导电材料（从溅射涂布机的金属）的均匀层，从而允许否则无法访问细节（ 图4G-I，5E）的回收。从湿的微环境，其中使用的未来样本良好质量的图像，以与电子电流被过充电，如果严重脱水和涂敷，可以实现（ 图4F，5A，5B）。重要的细节，如花粉壁表面（图5B，5C）和不同类型的indumenta（ 图5D-G）可以在深入研究不人工或不希望的颗粒或变形的形状。 <img alt="图4" src="/files/ftp_upload/55031/55031fig4.jpg" /> 图4: 早期（A，B），中（C-E） 和晚期（FI）花发育的SEM下拍摄。 （ 一 ） 塔拉 （莫利纳）Kuntze的总状花序与几个花分生组织。 （ 二 ）一个年轻的总状花序远志violacea Aubl的俯视图。 （ 三 ）Krameria的花芽ixine Loefl。在雌蕊群的分化。 （ 四 - 五 ） 刺桐芽藻。 （ 六 ）Krameria ixine的花的侧视图。花药和风格突出。 （G - I） 球兰 （L.）R·布朗。 （ <str在开花期翁&gt; G）花（与奥林巴斯SP-590UZ 26X拍摄数字图像）。 （H）的雄蕊和心皮裂顶视图。 （ 一 ）一对侧翼雄蕊心皮2的侧视图。星号表示雄蕊。 G =雌蕊。秤:（1，i）的1毫米，（B，D）为400μm，（C）为200μm，（E）500微米，（F，H）2毫米，（G）0.25厘米照片采取了MA贝洛。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55031/55031fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55031/55031fig5.jpg" /> 图5: 孢子囊和花粉的SEM照片（A，B），详细的表面（C-e） 和indumenta（F - 克）。 （ 一 ） 鳞毛蕨藻孢子囊。 樱桃椇 DA网络（ 二 ）花粉B目标上的耻辱。 （ 三 ） 翼状猪笼草布兰科的花粉。 灯盏的花序（ 四 ）侧视karvinskianus DC。图像拍摄的BSE检测选项。 （ 五 ）苔藓Peristoma SP的侧视图。 （ 六 ）腺和迷迭香 L的叶子的背面上没有腺毛。 （G） 油橄榄 L的叶平秤的顶视图。秤:（一）为400μm，（B）为60μm，（C）5微米，（d）第4毫米，（E）600微米，（F）200微米，（G）为600μm。照片是采取Y.鲁伊斯 - 莱昂。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55031/55031fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 不同基质对囊肿继发水霉寄生的脊柱生长的影响表1， 图6a）显示出环路。在碳和金网格刺较短，卷曲和周围的囊肿更旺盛的生长不会形成回路（ 图6B，6D）。虽然铜网上，刺倾向于卷曲不用循环，他们中的一些细长的（ 图6C）。在鱼的鳞片，刺人卷曲，表面周围丰富的，有时形成环端（ 图6E-F）。 <img alt="图6" src="/files/ftp_upload/55031/55031fig6.jpg" /> 图6: 水霉寄生 在浸在液体介质囊肿 棘微分的增长模式 。 （ 一 ）GLAS秒。 （ 二 ）碳。 （ 三 ）铜。 （D）黄金。 （ 五 ）哈克规模。 （ 六 ）鲑鱼的规模。直刺分别在玻璃和铜的发展，而形成碳，金，和鱼鳞卷曲棘。刺的钩头（白色箭头）是在玻璃上生长的所有刺并在鱼鳞几个刺观察到。囊肿壁是黄色。规模:20微米。照片是采取Y.鲁伊斯 - 莱昂和S. Rezinciuc。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55031/55031fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>表面</td><td>脊柱形态</td><td></td></tr><tr><td></td><td>伸长</td><td>环在尖端</td></tr><tr><td>玻璃盖</td><td>良好的延伸率，刺容易算</TD&gt; <td>当下</td></tr><tr><td>鱼鳞</td><td>卷发造型不容易刺算</td><td>当下</td></tr><tr><td>透射电镜网格</td><td>短而卷曲刺不容易算</td><td>缺席</td></tr></tbody></table> 表1:脊柱形态使用不同类型的衬底。 意外的观测Phellorinia herculanea F。 斯泰拉塔 除了乳汁菌丝上P. herculanea，德卡隆赫等的exoperidium的意外发现。 31发现球状，并与在该材料脱水CPD由于在微波进行再水化步骤孢子混合光滑护士细胞（8-13微米）。 （ 图1d）。结构和叔的壁的细节他护士细胞和孢子保存完好，尽管他们的差分组合物（ 图1C-D）。

Watch this Scientific Journal Video about Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples at JoVE.com

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

扫描电子显微镜（SEM）协议有问题的工厂，卵菌和真菌样本

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

scanning-electron-microscopy-sem-protocols-for-problematic-plant

Nanyang Technological University

Research

JoVE Journal

Biology

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

28.7K 次观看.  Real Jardín Botánico, CSIC. Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here. 

视频: Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

科研

生物学

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

自噬的分析<em&gt;产黄青霉</em&gt;通过结合使用饥饿垫荧光显微镜

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

一个标准化的方法肝窦内皮细胞的分析及其窗孔用扫描电子显微镜

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

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 ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (&#62;100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.

Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.

通过低温扫描电子显微镜研究光合膜内冻结，割断叶片组织的超分子组织

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

准备厚生物样品的观察扫描透射电子断层扫描

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

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 ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

阵列断层扫描工作流程，用于使用扫描电子显微镜有针对性地采集体积信息

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...