This presentation was supported by a Clinical Innovator Award to JLC from the Flight Attendant Medical Research Institute and by the United States Environmental Protection Agency. Although the research described in this article has been funded wholly or in part by the United States Environmental Protection Agency through Cooperative Agreement CR83346301 with the Center for Environmental Medicine, Asthma, and Lung Biology at The University of North Carolina at Chapel Hill, it has not been subjected to the Agency&#8217;s required peer and policy review, and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

1，制备生物样品的冷冻断裂/冷冻蚀刻<ol><li>使用常规EM固定剂配方如2％戊二醛+ 2％多聚甲醛的0.1M磷酸盐缓冲液1小时。进行生物体组织的主要固定。注意:虽然它可能是可取的，冻结某些类型的样品无需事先固定的，普遍血液传播病原体的预防措施强制适当的固着，其中试样由人体组织。 </li><li>以下主要定影，冲洗样品在补充有0.2M的蔗糖不超过1小时，在相同的缓冲液。 </li><li>样品转移到在0.1M磷酸盐缓冲液组成的25％甘油的冷冻保护剂溶液为不超过1小时以上。 </li></ol> 2，冷冻和标本的存放在冷冻断裂的研究进展<ol><li>的适当大小和形状的试样选择金或铜试样存根被处理（ 图4中 </st荣&gt;）。 </li><li>使用细级镊子和/或在金属试样存根顶部的试样的小轨距的注射器针头位置的小片段。 <ol><li>减少样品的液体含量为粘性的，略微粘状稠度通过从用滤纸短截线的边缘处撤出液体。 </li><li>对于单副本，用小规格的注射器针头上的存根创建组织的土丘。对于双副本，反转另一桩和位置也正好在存根，轻轻放置到样品表面创建一个三明治（ 图5A）。不要按样品从两个桩之间。 </li></ol></li><li>填写两根绝缘的杜瓦瓶液氮。注:第一艘可容纳含小灵通是用来从市售缸（ 图5B）液化少量丙烷气的金属柱。第二个容器是一个分区的存储/保持器的简要LY保持在液氮下冷冻样品。 <ol><li>使用定位在丙烷以及气缸喷嘴，打开气瓶阀和使气体流入所述第一容器中，其中，将液化的阱。注意:小心！丙烷爆炸，可能会造成冻害开接触，是一种窒息。仅在化学罩用于这种用途照顾也避免了外来火源，用衣服保护防寒，保持足够的通风认证使用丙烷。 </li><li>尽可能快地，拿起试样安装与细级镊子和沉浸入液化气体的第一容器几秒钟，然后很快转移到液氮中的第二保持器（ 图5B）。 </li><li>一旦样品被冻结，液态氮，直到准备下存储转移到冷冻断裂装置。存根转移到一个更大的存储液氮杜瓦容器扩展如果需要的话，但是关心存放时，应注意不要让存根解冻。 </li></ol></li></ol>冷冻断裂3。操作仪器和样品处理<ol><li>在黄铜保持器并进入腔室中的标本<ol><li>加载金试样存根成小册子型双复制品试样保持器或液氮下夹紧装置和保持在那里，直到准备在试样腔室（ 图6）来定位。 </li><li>当腔室已达到高真空（约2×10 -6毫巴）及阶段已被冷却至液氮温度，则关闭泵，排气腔室，并尽可能快地定位安装试样存根到样品表。 </li><li>启动泵，重新建立在高真空，并使用电子级和手臂温度控制来调节样品台温度为-100℃，并直接液氮到切片机臂和BRI它纳克至液氮温度。 </li></ol></li><li>的断裂过程 <ol><li>当在液氮温度下实现高的真空度，-100ºC阶段的温度和切片机臂，远程打开双复制品试样保持器，从而压裂在试样架的标本（冷冻fracture_movie1.mov）。 </li><li>在标本用于蚀刻下骨折的情况下，使用保持在夹紧刀片在切片机臂刮（ 即骨折）的标本，以代替步骤3.2.1表面（冷冻etch_movie2.mov）。 </li><li>如果添加的冷冻蚀刻步骤将被执行，放置冷却，切片机臂在断裂面为一至数分钟。注意:此动作会升华（ 即蚀刻）水从断裂面。这个动作的效果是揭示真实细胞表面，细胞外基质，和/或分子组装的水升华，除了FRActure面孔穿过细胞膜的脂双层。 </li></ol></li><li>金属遮蔽和副本支持蒸发用电子枪 <ol><li>激活的铂/碳的电子或电阻枪，并允许它从30度的角度蒸发一薄层铂/碳（约2纳米）以上的断裂面 - 45°。这通常需要大约15 - 20秒。 </li><li>激活的碳电子或电阻枪如步骤3.3.1和蒸发一层薄薄的碳的断裂试样表面的直接开销（90度）给予副本的支持。这通常需要大约15 - 20秒。 </li></ol></li><li>副本检索<ol><li>当复制阴影和碳稳定后，关闭真空泵，发泄室，取出试样从仪器安装。 </li><li>使用一对细粒级镊子从双删除的每个黄金标本存根副本小册子/夹，并允许前，轻轻地降低存根到水面上的斑点板（ 图7）解冻几秒钟。注:碳/铂副本包含附着的样品材料会浮到水面。 </li><li>操纵使用一个细金属丝环，或通过细级镊子和转移到另一个点板含5％重铬酸钠在50％硫酸为一至数小时保持以往的铜电子显微镜网格中的副本。注:此浴从副本消化的实际生物样品材料。充分强度的家用漂白剂可被取代为重铬酸盐/硫酸溶液。 </li><li>当消化完成后，传送回副本到一个干净的水面，检索到一个标准的铜电子显微镜网格。 （注:副本可​​能会碎裂成小块，在消化过程中，但即使是非常小的副本可能包含大量的信息，并应RETRieved和检查。）店铺副本在市售格箱，他们将保持稳定多年，柔和处理配套电网。 </li></ol></li></ol>冷冻断裂/蚀刻副本4超微结构检查<ol><li> 80千伏 - 视图中的透射电子显微镜在加速电压通常为50复制品。 </li><li>记录在标准的TEM薄膜或用高分辨率的数码相机相关的图片。 </li></ol>

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The author has nothing to disclose.

在下面的介绍和商业可用性的岁月，冷冻断裂/蚀刻方法被广泛用于生物膜结构的调查。事实上，一些膜的结构特化的最佳角度已在冷冻断裂/蚀刻制剂获得。这些研究不仅有助于细胞膜的结构组织的了解也深入介绍了如何结构和功能有关。 常规生物电子显微镜的出现所带来的认识，与醛固定剂，反应性化学物质，并赋予差分电子密度的生物标本所需的重金属的化学处理本身对...

这个概念和生物标本冷冻断裂处理实际应用一个世纪前引入了斯蒂尔1半以上。早期的设备，不同的拨款组件集成到一个工作独立单元1。原装置进行了修改，并精制成商业上可用的工具，以容纳用于远程操作，维修高真空的迫切需要，并且碳和金属的蒸发，以产生由透射电子显微镜（ 图1和图适合于考试的复制品2）。 典型的仪器由一个高真空室与样品表和（ 图1）切片机臂具有可调节的液氮通过量。该腔室还容纳两个电子枪，一个用于稳定碳蒸发定位在90度角到试样阶段，另一个用于普拉蒂NUM /碳遮蔽在一个可调节的角度，典型地15° - 45°（ 图2）。本机的电源被应用到操作真空泵及电子面板调节温度调节和电子枪控制。 最初的设想为手段，以实现病毒改进成像，冷冻断裂甚至获得了更多的人气，作为参加考试和细胞膜的分析和自己的专业2,3的技术。事实上，此过程一直积分来阐明细胞和组织中，许多这些研究站，以细胞和分子生物学4-9经典捐款的结构/功能关系。用于冷冻断裂技术的发展的主要目标和理由是限制工件可观察到的，在电子显微镜的分辨率从化学固定和加工中常规生物电镜用于导出。这里的目标是限制化学固定和冷冻以足够的速度和频繁的冷冻保护剂，以限制冰晶形成和其它冷冻的工件存在检体。最近，这一技术已经发现了从分子生物学家和材料科学的调查审核纳米粒子和纳米材料的兴趣死灰复燃。 冷冻断裂和冷冻蚀刻图像呈现出立体字，有时被误认为是扫描电子显微照片。然而，冷冻断裂制剂通过透射电子显微镜观察和高分辨率的形态学研究的主要贡献是细胞膜的结构/功能元件的独特表示。冷冻断裂处理是通过冷冻的细胞和组织具有足够的速度以限制冰结晶和/或与使用冷冻保护剂如甘油引发。试样，然后在真空和代理商断裂LICA是由碳和铂在断裂面的蒸发产生的。最初的样本是从被检索到一个标准的EM试件格子副本消化。冷冻断裂图像的另一种常见的误解是，它们显示了细胞表面上。然而冷冻断裂的基本前提是，生物膜穿过脂双层的破裂过程（ 图3）分离。这个过程在生物膜产生2断裂面，其中一个显示邻近于细胞质中，PF-面在膜的一半的组织，并且其中一个显示在膜的双分子小叶的一半，相邻的外氛围中，EF面。真细胞表面没有在冷冻断裂图像表示，但只有当冷冻蚀刻后的断裂过程的后续步骤中加入用于显示。为了有效地刻蚀先前断裂试样以显示表面德泰L，样品必须以极快的速度，没有unetchablecryoprotectant被冻结。从断裂试样揭示标的特征的表面蚀刻的水被定位在冷却切片机臂在样品阶段创建阶段之间的温度差保持在检体和所述冷却切片机臂，导致​​水从表面升华来实现的。当水从断裂试样的表面上的冷冻蚀刻机动过程中升华，然后实际细胞表面，细胞外基质，细胞骨架结构和分子组装的各方面可以以高分辨率显示。因而冷冻断裂和冷冻刻蚀不能互换术语而是反映逐步的过程，其中后者可以不依赖于特定研究的需要是必须的或可取的。 以下的冷冻断裂/冷冻蚀刻程序，断裂表面进行定向evapora略去碳和铂的外套，以提供支撑和成像对比的复制品。铂/碳成像蒸发可以是单向的或旋转​​，并通过任一电阻或电子枪来实现的。从已知的角度，一般30度单向阴影 - 45度，是在执行某些形态的计算是有用的。已进行深蚀刻试样通常是旋转遮挡和这些样品的所得图像评价是照相反转。 的历史以及在冷冻断裂/冷冻蚀刻技术的当前目标是限制化学固定和处理样品是与更常规的透射式电子显微镜的程序相关联的伪影。然而，这种技术提供了在其赋予三维细节，从而促进采集生物，材料科学形态的数据，并且n的能力的实质性优点anotechnology标本。冷冻断裂和冷冻蚀刻过程是复杂的，多方面的，其应用的某些方面进行定制。此演示文稿提供了进程的主要特点的调查来看，读者被称为全面的出版协议10，11，以解决细节和定制过程中对特定的研究需求。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Balzers Freeze-fracture/freeze-etch plant</td>
			<td style="width:71px;">Balzers</td>
			<td style="width:119px;">BAF400T</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard buffers</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">standard aldehyde fixatives</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sodium dichromate</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sulfuric acid</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Disposable supplies for Platinum/Carbon Evaporation</td>
			<td style="width:71px;">Technotrade International</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Liquid nitrogen</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Freon</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Disposable supplies for electron microscopy</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transmission electron microscope</td>
			<td style="width:71px;">Carl Zeiss Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

fundamental technical elements of freeze-fracture/freeze-etch in biological electron microscopy

冷冻断裂/冷冻刻蚀描述由此标本，通常在自然界中的生物或纳米材料，冷冻，破碎，和复制，以产生碳/铂"铸造"用于检查通过透射电子显微镜的方法。标本进行超速冷冻速率，往往在冷冻保护剂的存在下，以限制冰晶形成，与样品的后续压裂在液氮冷却的高真空条件下的温度。所得到的断裂面进行复制，并从该表面赋予立体细节来铸造一个角度稳定的由碳和铂的蒸发。这种技术已被证明为细胞膜和其专业的调查特别有启发性，并大大的细胞形态相关细胞功能的理解做出了贡献。在这份报告中，我们调查了仪器的要求和技术协议执行冷冻断裂时，相关的命名法和断裂面的特征，变型的常规方法，和标准的冷冻断裂解读图像。这种技术已被广泛用于细胞生物学的许多领域超微结构的调查和有希望的分子，纳米技术的新兴成像技术和材料科学的研究。

冷冻断裂图像判读的关键前提是，断裂面穿过膜赋予2断裂面的脂质双层，称为按约定的PF-面（等离子断裂面）和EF-面（外断裂面）（ 图3）。的PF-面是相邻的小区和EF-面的细胞质中的膜脂双层的一半是邻近于细胞外环境的膜脂双层的一半。该冷冻断裂技术是膜结构的调查是特别有用的，并在两个表面都典型地与PF-面孔被填充在对比EF-面含有更少的许多膜相关联的颗粒明显不同。冷冻?...

Watch this Scientific Journal Video about Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy at JoVE.com

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

基本的技术和用于检查通过透射电子显微镜生物标本和纳米材料的冷冻断裂加工的精炼进行说明。这种技术对于揭示超微结构特点和生物膜的专业和获得超微结构水平尺寸和空间数据在材料科学和纳米技术产品的首选方法。

冷冻断裂/冷冻蚀刻的生物电子显微技术的基本要素

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

fundamental-technical-elements-freeze-fracturefreeze-etch-biological

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

33.1K 次观看.  The University of North Carolina at Chapel Hill. 基本的技术和用于检查通过透射电子显微镜生物标本和纳米材料的冷冻断裂加工的精炼进行说明。这种技术对于揭示超微结构特点和生物膜的专业和获得超微结构水平尺寸和空间数据在材料科学和纳米技术产品的首选方法。 

视频: Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

科研

生物学

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

裂殖酵母性生命周期的显微镜

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

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 ...

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 ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

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, ...

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 ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.

冷冻断裂/冷冻蚀刻的生物电子显微技术的基本要素

摘要

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电子显微镜对胚胎的制备果蝇胚胎心管

时间推移显微镜早期胚胎发育线虫

裂殖酵母性生命周期的显微镜

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术

准备厚生物样品的观察扫描透射电子断层扫描

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

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

生物组织样本的串行块面扫描电子显微镜（SBF-SEM）

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

优化低温电子断层扫描数据采集策略

冷冻断裂/冷冻蚀刻的生物电子显微技术的基本要素

摘要

探索更多视频

此视频中的章节

电子显微镜对胚胎的制备果蝇胚胎心管

时间推移显微镜早期胚胎发育线虫

裂殖酵母性生命周期的显微镜

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术

准备厚生物样品的观察扫描透射电子断层扫描

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

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

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

优化低温电子断层扫描数据采集策略

生物组织样本的串行块面扫描电子显微镜（SBF-SEM）