我们衷心感谢茂雄的宝贵建议和讨论。我们还感谢约翰和 Sumire Eckstein 对手稿的批判性阅读。

注: 本研究中使用的标本为微生物, 在液氮中迅速冷冻, 用丙烷取代含有2% 锇氧化的丙酮, 并嵌入环氧树脂1,2,3, 4,5,6,7,8,9,10,11,12,13 ,14,15,16,17,18。
1. 支持膜的制备 (图 1-3)
注: 使用 cast-on-glass 方法19准备了银色的 Formvar 支持胶片。
<ol><li>在二氯化乙烯中加入100毫升 1.5% Formvar 的 Formvar 制造装置 (图 1)。通过用橡皮球19将溶液与空气通过 "a", 在该装置的上部柱上的 Formvar 溶液中浸 one-half (76 mm x 26 mm x 1.3 mm)。<ol>
<li>通过打开三路塞并通过 "b" 释放空气以减少压力, 从柱上排出溶液。取出装置上的玻璃片, 在空气中晾干, 在玻璃滑梯的表面形成薄膜。使用白炽灯加速干燥的 Formvar 膜。</li>
	</ol>
</li>
	<li>在用刀片 (图 2a) 将玻璃片上的四边缘刮掉后, 并在幻灯片上呼吸以方便胶片从幻灯片20中分离出来, 将玻璃片浸入水中, 使胶片漂浮在水缓慢地在低水平角度 (约10°,图 2b)。</li>
	<li>用铝架 (30 mm x 25 mm x 3 mm) 用孔 (直径 4 mm) 舀出 Formvar 膜 (图 3a)。将机架与 Formvar 胶片放在干燥中直到使用 (图 3b)。</li>
</ol>2. 在显微镜 (图 4) 下, 用超声波修剪刀片和刀片式服务器21修剪试样块
<ol><li>通过用光学显微镜 (图 4a) 观察块的尖端, 确保块表面有细胞。</li>
	<li>将试样块装入卡盘 (块座), 然后在修剪阶段安装卡盘21 (图 4b)。这种修剪阶段有一个从标本背面的照明机制。</li>
	<li>修剪块的大小为 0.7 mm x 1.0 mm (图 4c, 5d)。由于环氧树脂是非常硬, 修剪的方块首先使用超声波修剪刀片。超声波修边刀是一种新引进的机器, 其块体易于修剪。然后用剃刀刀片进一步修剪木块。剪切一个肩膀以标记切削方向 (请参见图 5d,图 8)。</li>
</ol>3. 使用切片用金刚石刀修整试样块 (图 5)
<ol><li>在 ultramicrotome 标本夹中设置试样块夹头。在执行连续超薄切片时, 将试样的90°逆时针放置到实际位置 (请参见图 5d)。</li>
	<li>用菱形修剪刀切割块的表面 (参见图 5d)。将菱形刀边与试样块面平行, 并切下试样表面的最小量, 以免丢失任何标本 (图 5d)。 注意: 这一步是做抚平的标本表面。细长部分的试样表面部分保持完好 (因此, 这部分不像镜子那样闪亮,图 5d), 这样就不会丢失样本的任何部分进行连续切片。这是因为标本是暴露在表面的块。</li>
	<li>在刀台上放置一面镜子 (台面切割, M), 以监视试样块的切割 (图 5c)。</li>
	<li>切割左边缘 (这成为上侧,图 5d, 在序列切片的样本) 的块使用修剪刀旋转刀阶段30°向左 (图 5a)。</li>
	<li>从试样的左边缘切下100µm 的块面 (这成为序列切片中试样的下部,图 5d), 通过将刀阶段30°向右旋转 (图 5b)。 注: 该系列的部分将削减约 90 nm x 约1毫米大小的标本薄部分。大的部分将用于调整试样表面和刀口, 使其平行。在将试样表面调整到与刀口平行的地方后, 将用刀片将大零件移除。通过使用切片切割试样的上下两侧, 试样的两侧变得平滑且完全平行 (图 5d), 这是获得平直和完整的带状剖面所必需的。</li>
</ol>4. 调整试样表面和刀口, 使其与其他21平行
<ol><li>取下裁切刀, 顺时针旋转试样块90°。</li>
	<li>把超薄切片刀放到刀台上</li>
	<li>调整试样表面和刀口, 使其与试样表面的大部分面平行。 注: 试样的大部分用于调整, 因为连续切片的切割面很小, 很难用这部分来调整试样表面和刀口, 特别是在垂直方向。因此, 使用的大部分标本, 使调整容易。</li>
</ol>5. 在试样块上散布氯丁橡胶溶液 (图 6)
<ol><li>要将标本夹头放置在与原始位置完全相同的位置, 请在卡盘和切片的卡盘上应用磁带, 并在边界处剪切 (图 6a)。</li>
	<li>从切片取出标本夹头, 并将其置于显微镜 (图 6b) 下。用剃刀刀片切断标本的大部分, 留下纤细的部分, 从中可以获得串行部分。</li>
	<li>使用巴斯德吸管, 下降约1µL 0.5% 氯丁橡胶解决方案 (图 6b) 上的标本, 以用于串行切片, 使节方胶粘剂。用氯丁橡胶溶液覆盖整个试样。立即吸收过剩的氯丁橡胶解决方案与一块滤纸放置在靠近标本。获得一条丝带的部分是必不可少的拍照的串行细胞部分, 和氯丁橡胶胶水是非常有用的, 以坚持节。</li>
	<li>准备3狭缝网格 (狭缝尺寸: 中间, 0.4 毫米 x 2.2 毫米, 两侧 0.2 mm x 2.2 mm) (图 6c) 通过将网格的手柄弯曲到60°, 用0.5% 氯丁橡胶溶液处理网格, 并使网格亲水性辉光放电22。</li>
</ol>6. 制作串行部分 (图 7-9)
<ol><li>将样本块卡盘放回切片, 并将其与 5.1 (图 6a) 对齐, 使其与录音部件对准。这使块面和刀边完全平行。然后把刀靠近标本。</li>
	<li>把刀船装满水。</li>
	<li>用塑料盖盖住切片以防止气流 (图 7)。 注: 在超薄切片和剖面提取过程中的气流通常会引起问题。塑料盖有三孔: 一个孔是为双目透镜, 另外两个孔是为胳膊允许操作, 当盖子是。木扶手是用来放置武器, 而做微妙的工作, 如检索串行部分与网格。木扶手被放置在不同的表从切片表 (图 7, 箭头), 以便不传送操作员的手的振动到切片。</li>
	<li>开始切割 200 nm 截面厚度的试样 (图 8)。设置在 200 nm 厚度将节省时间把刀的表面上的标本。</li>
	<li>剪切第一个截面后, 将截面厚度设置为 70 nm (图 8)。</li>
	<li>当连续区段的数量到达 20 (确切地说, 在丝带以后到达大约1.8 毫米; 区段的数量根据区段的宽度变化), 设置部分厚度到10毫微米 (图 8), 当继续切开。 注: 由于切片不能切割10纳米厚的截面, 没有新的节出现, 和以前的切割部分成为分离的刀口。重要的是要获得分离的节组 (图 9) 来拾取串行部分, 以避免使用一对头发线手动分离分区的困难。因为视野在传输电子显微镜是2.0 毫米, 部分应该是1.8 毫米长至多。</li>
	<li>将截面厚度设置为 60 nm (图 8)。这将生成 70 nm 剖面 (图 8), 因为该计算机将添加以前的 10 nm 厚度。</li>
	<li>将截面厚度设置回 70 nm (图 8), 并继续切削, 直到获得 1.8 mm 长的剖面。</li>
	<li>重复 6.6-6.8, 直到五 1.8 mm 长剖面得到 (图 9)。我们通常做五节组, 因为有五标本持有人在透射电子显微镜可在我们的实验室, 但五不是极限。</li>
</ol>7. 拾取串行部分 (图 10-12)
<ol><li>将 "节保持环路"23 (循环的内径: 5.0 mm) (图 10a) 放在第三个分区组 (图 10b) 上。 注意: 有必要按其切片顺序检索各节以按正确顺序拍摄图片。然而, 由于在刀船上有五节组, 它往往会混淆哪些群体是哪一组。通过将循环放在第三个组上, 就可以清楚地看出, 运算符附近的两个组是第一和第二类, 而从运算符到距离的两个群是第四和第五组 (图 10b)。这将防止混合订单。</li>
	<li>将 "水面提升回路"23 (WSRL, 循环的内径: 4.0 mm) (图 11a) 放在刀台上 (图 11b)。WSRL 将坚定地站在刀台上, 因为它在底部装有磁铁。<ol>
<li>通过移动轴和手柄, 将 WSRL 的环路放在串行节的上方。把 WSRL 的环放在水面上, 这样循环就会包围这些部分。</li>
		<li>通过将螺钉向下转动来推动循环, 使水面表面张力升高 (图 11 c-d)。将剖面移动到循环的中心, 将其放置在水的顶点上, 并使用发束21调整剖面的方向。</li>
		<li>拿起部分组通过触摸它与一个裸露的狭缝网格, 这是由镊子举行, 并保持平行的水面 (图 11d)。对于部分来说, 首先接触网格 (而不是水), 将剖面精确地放置在网格的中心 (图 11d) 是很重要的。</li>
	</ol>
</li>
	<li>将网格与剖面一起放置在 Formvar 支持膜上的小水滴16 (图 12), 并使用滤纸去除多余的水。 注: 由于膜 (切片) 直接触及膜 (支撑膜), 支撑膜的皱起通常是一个问题。通过在 Formvar 支持膜上放置一个带截面的网格和少量的水来减少支撑膜上的皱纹的问题,16。</li>
</ol>8. 着色部分16,24 (图 13)
<ol><li>在切片完全干燥后, 将 Formvar 膜从网格周围撕裂, 并取下带有剖面的网格。</li>
	<li>在染色管的凹槽中设置网格16 , 按正确的顺序 (图 13), 并将铀醋酸盐和柠檬酸铅24的着色切片。 注: 有一个沟槽0.6 毫米深沿长轴的管切割与刀片。该管用于防止在使用镊子时的辅助薄膜发生意外破损, 因为在染色和洗涤过程中, 不需要直接处理网格,16。</li>
</ol>9. 对串行部分的观察 (图 14)
<ol><li>按照正确的顺序 (图 14a) 将 multi-specimen 托架中的5网格设置为垂直于试样夹轴的网格狭缝的长轴。网格的手柄 (箭头) 不必被切割, 因为 "网格毒贩" 不会触及手柄。</li>
	<li>用 "网格毒贩" (图 14b) 固定网格, 并将标本夹插入透射电子显微镜。 注意: 使用所有单元格部分 (图 15) 的低放大图像通常是有用的, 以便在拍摄目标单元格之前, 在没有污染和良好的固定状态下找到有趣的微生物或细胞。</li>
	<li>在高倍率下拍摄所有单元格中目标单元格的图片。</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Support+films.">Support films.</a> Principles and Techniques of Electron Microscopy: Biological Application. , 216 (2000).</li><li>Yamaguchi, M., Kita, S., The Japanese Society of Microscopy, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin+sectioning.">Ultrathin sectioning.</a> Guide Book for Electron Microscopy. , 40-52 (2011).</li><li>Yamaguchi, M., Aoyama, T., Yamada, N., Chibana, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+hydrophilicity/hydrophobicity+of+the+plasma-polymerized+naphthalene+film+(Super+support+film)+and+other+support+films+and+grids+in+electron+microscopy.">Quantitative measurement of hydrophilicity/hydrophobicity of the plasma-polymerized naphthalene film (Super support film) and other support films and grids in electron microscopy.</a> Microscopy. 65, 444-450 (2016).</li><li>Kita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+method+for+obtaining+serial+ultrathin+sections+using+new+small+tools.">Reliable method for obtaining serial ultrathin sections using new small tools.</a> Kenbikyo. 46, 253-257 (2011).</li><li>Yamaguchi, M., Shimizu, M., Yamaguchi, T., Ohkusu, M., Kawamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+use+of+uranyl+acetate+solution+in+section+staining+in+transmission+electron+microscopy.">Repeated use of uranyl acetate solution in section staining in transmission electron microscopy.</a> Plant Morphology. 17, 57-59 (2005).</li><li>Hayworth, K. J., Kasthuri, N., Schalek, R., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automating+the+collection+of+ultrathin+serial+sections+for+large+volume+TEM+reconstructions.">Automating the collection of ultrathin serial sections for large volume TEM reconstructions.</a> Microsc. Microanal. 12, 86-87 (2006).</li><li>Hildebrand, D. G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-brain+serial-section+electron+microscopy+in+larval+zebrafish.">Whole-brain serial-section electron microscopy in larval zebrafish.</a> Nature. 545, 345-349 (2017).</li><li>Denk, W., Horstmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+block-face+scanning+electron+microscopy+to+reconstruct+three-dimensional+tissue+nanostructure.">Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.</a> PLoS Biol. 2, e329 (2004).</li><li>Heymann, J. A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+3D+imaging+of+cells+and+tissues+with+a+dual+beam+microscope.">Site-specific 3D imaging of cells and tissues with a dual beam microscope.</a> J. Struct. Biol. 155, 63-73 (2006).</li></ol>

作者没有什么可透露的。

这里提出的方法不需要昂贵的设备。它只需要一个铝架 (图 3)、三狭缝网格 (图 6c)、节保持环路 (图 10a)、water-surface 提升环路 (图11a) 和染色管 (图 13)。目前的方法有许多特点。试样的大部分用于调整试样的表面和刀口, 使其彼此平行。在将一组串行剖面检索到狭缝网格时, 将序列切片分成组, 以避免使用一对头发线分隔节的困难。"节保持环" 用于避免混合节组的顺序。"水表面提升回路" 用于确保剖面位于水的顶端, 并且首先接触网格, 以便将它们放置在栅格上的所需位置。铝架上的 Formvar 支撑膜用于使网格上的剖面恢复更容易, 并避免支撑膜起皱。采用染色管, 避免用镊子意外折断支撑膜。
最近, 一个自动的磁带收集 ultramicrotome 被开发了25 , 以收集部分自动扫描电子显微镜 (SEM) 成像的电子不透明的塑料磁带。此方法可以可靠地切割数千个超薄部分26, 这是目前方法所无法实现的。此外, 串行块面 (SBF) 27和聚焦离子束 (纤维蛋白原)-SEM28用于收集动物细胞和组织的3D 信息。在这些方法中, 用金刚石刀或聚焦离子束进行切片是在 SEM 显微镜下集成的, 并以完全自动的方式进行, 无需特殊技术。虽然这些方法是有用的, 设备是如此昂贵, 没有许多机构购买这些机器。此外, 由于这些方法采用扫描电镜的照片部分, 显微的分辨率低于目前的方法, 采用透射电子显微镜 (TEM)。这些方法目前无法解决单个核糖体微粒、微管、丝和 DNA 纤维。
对于微生物的 structome 分析和未知微生物的超微结构研究, 有必要观察细胞壁、细胞膜、线粒体、细胞核、细胞核膜、内质网、叶绿体, 质, 高尔基体仪器, 核糖体, 和丝状元素在较高的分辨率。连续超薄切片的 TEM 观察是在三维度中进行这种分析的几种方法之一。由于超薄切片在观察后保持不变, 可以重复观察同一区域;这不是可能的在 SBF 或说谎-SEM, 因为被观察的区域在观察以后丢失。这一特性对于研究深海微生物是很重要的, 在这种情况下, 必须先在低放大率下搜索有趣的生物体, 然后在高放大倍数下以连续的目标生物体进行拍照。
总之, 目前的研究可以简单、廉价、可靠的方式进行连续超薄切片, 这对电子显微镜下高分辨率微生物的 3D ultrastructual 研究至关重要。

适当的连续超薄切片技术是必不可少的研究细胞和细胞成分三维在电子显微镜水平。研究了酵母细胞周期中纺锤极体的动态变化, 并揭示了其在细胞周期和重复时间上的形态学改变1,2,3, 4,5。在 2006年, 我们创造了一个新词 ' structome ' 通过结合 "结构" 和 "-", 并定义为 ' 定量和三维结构信息的整个细胞在电子显微镜水平 ' 6,7。
通过 structome 分析, 需要连续超薄切片技术, 发现酵母细胞的酿酒酵母和外 dermatitidis有大约20万核糖体7,8, 一个大肠杆菌细胞有2.6万核糖体9, a 结核分枝杆菌细胞有1700核糖体10 , Myojin 螺旋菌只有300核糖体11。这些信息不仅可以用来估计每个生物体的生长速率, 也有助于识别物种的9。
此外, structome 分析导致发现一个新的有机体;Parakaryon myojinensis在日本海岸的深海中发现, 其细胞结构介于原和真核之间的中间,121314,15。目前, 连续超薄切片技术被认为是非常困难的, 需要很长时间才能掌握。在这项研究中, 我们开发了一个可靠的方法, 任何人都可以进行连续超薄切片无困难。

<table>
	<tbody>
		<tr>
			<td>Formvar making apparatus</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>652</td>
			<td>W 180 x D 180 x H 300 mm</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Matsunami Co. Ltd., Osaka</td>
			<td>-</td>
			<td>76 x 26 x 1.3 mm</td>
		</tr>
		<tr>
			<td>Aluminum rack with 4-mm holes</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>658</td>
			<td>W 30 x D 25 x H 3 mm, Refer to this paper</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>SMZ 645</td>
		</tr>
		<tr>
			<td>LED illumination for stereomicroscope</td>
			<td>Nikon Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>SM-LW 61 Ji</td>
		</tr>
		<tr>
			<td>Trimming stage</td>
			<td>Sunmag Co.Ltd., Tokyo</td>
			<td>-</td>
			<td>Tilting mechanism equipped, Refer to this paper</td>
		</tr>
		<tr>
			<td>LED illumination for trimming stage</td>
			<td>Sunmag Co.Ltd., Tokyo</td>
			<td>-</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Ultrasonic trimming blade</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>5240</td>
			<td>EM-240, Refer to this paper</td>
		</tr>
		<tr>
			<td>Diamond knife for trimming</td>
			<td>Diatome Co. Ltd., Switzerland</td>
			<td>-</td>
			<td>45&#176;</td>
		</tr>
		<tr>
			<td>Diamond knife for ultrathin sectioning</td>
			<td>Diatome Co. Ltd., Switzerland</td>
			<td>-</td>
			<td>45&#176;</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica Microsystems, Vienna</td>
			<td>-</td>
			<td>Ultracut S</td>
		</tr>
		<tr>
			<td>Mesa cut</td>
			<td>Leica Microsystems, Vienna</td>
			<td>-</td>
			<td>Mirror</td>
		</tr>
		<tr>
			<td>0.5% Neoprene W solution</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>605</td>
			<td></td>
		</tr>
		<tr>
			<td>Special 3-slit nickel grid</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>2458</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Special 3-slit copper grid</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>2459</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Section-holding loop</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>526</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Water-surface-raising loop</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>527</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Staining tube</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>463</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Multi-specimen holder</td>
			<td>JEOL Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>EM-11170</td>
		</tr>
		<tr>
			<td>JEM-1400</td>
			<td>JEOL Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>Transmission electron microscope</td>
		</tr>
	</tbody>
</table>

a method for obtaining serial ultrathin sections of microorganisms in transmission electron microscopy

在透射电镜高倍率下观察三维细胞和细胞成分, 需要制备试样的超薄切片。虽然准备连续超薄的部分被认为是非常困难的, 它是相当容易的, 如果正确的方法是使用。本文介绍了一种安全获取微生物连续超薄切片的 step-by 步骤。该方法的要点是: 1) 使用试样的大部分, 调整试样的表面和刀口, 使其相互平行;2) 在狭缝网格上检索一组连续剖面时, 要在组中切割连续节, 避免在分离时使用一对头发束的困难;3) 使用 "节-保持回路", 避免混淆节群的顺序;4) 使用 "水表面提升回路", 并确保剖面位于水的顶端, 并首先接触网格, 以便将它们放置在栅格上所需的位置;5) 使用铝架上的支撑膜, 使其更容易恢复网格上的剖面, 避免支撑膜起皱;6) 使用染色管, 避免用镊子意外折断支撑膜。这种新的方法可以毫不费力地获得连续的超薄切片。该方法可以对3D 高分辨率的微生物细胞结构进行分析, 使用自动胶带收集 ultramicrotome 法和连续块面或聚焦离子束扫描电子显微镜是无法实现的。

在该协议中, 三-狭缝网格用于拾取串行段。网格是由镍或铜制成的。串行部分放置在中间狭缝上。双方的狭缝是必要的, 以查看的部分时, 他们拿起网格。在用镊子 (图 11d) 拾取时, 为了使网格与串行剖面保持平行, 手柄弯曲 (图 6c, 右)。一个小手柄有利于防止网格主体的弯曲, 在拾取时会引起严重的问题, 并防止在染色过程中掉节。该网格优于传统的 2.0 mm x 1.0 mm 孔网格。也就是说, 剖面由两个 0.4 mm 的金属棒直接支撑 (窄于1.0 毫米), 在三狭缝网格中由 Formvar 支持膜支撑, 而剖面仅由传统孔网格中的支撑膜支撑。因此, 使用三狭缝网格进行高倍率照相时, 可以防止试样漂移。
图 15显示了带有串行部分的五网格的低放大率视图。由于每个截面都粘在一起, 所以在更高的放大倍数下, 在下一节中很容易找到相同的单元格。图 16和17是单元格部分的序列图像的示例。由于在3狭缝网格上安全地支持节, 在高放大倍数 (x 5万) 上的照片很容易在没有试样漂移的情况下拍摄。脱氧核糖核酸2毫微米的纤维在这些研究被拍摄了9,12。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56235/56235fig1.jpg"> 图 1.Formvar 支持薄膜制作设备.在该装置的上部柱上, 有一半的玻片被浸在 Formvar 溶液中, 通过用橡皮球通过 "a" 来压出空气溶液。通过打开三路塞并通过 "b" 释放空气以减少压力, 解决方案从列中排出。(转载自山口和安达, 201119具有权限)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56235/56235fig2.jpg"> 图 2.从水中的玻璃滑梯上释放胶片的方法.(a) 玻璃片上的四边缘用刀片刮掉。(b) Formvar 膜在水上漂浮, 低角度缓慢降低玻璃滑动。(转载自山口和安达, 201119具有权限)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56235/56235fig3.jpg"> 图 3.Formvar 支持胶片在铝架上.(a) Formvar 膜从水中舀出铝架。(b) 机架存放在干燥, 直至使用。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56235/56235fig4v2.jpg"> 图 4.在显微镜下, 用超声波修剪刀片和刀片修剪试样块.(a) 在光学显微镜下观察标本块, 证实标本的存在。图中所示的块包含了与从深海收集的 chaeta (长度约1.7 毫米) 相关的微生物12,14。(b) 修剪阶段。(c) 显微镜的整体形象。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56235/56235fig5.jpg"> 图 5.用金刚石刀修整试样块切片.(a) 切割左边缘的图示。(b) 块面从试样的左边缘处切割约 100 nm 的位置。(c) 放置在刀台上的镜子 (台面切割, M)。(d) 试样表面。注意, 细长部分的上侧和下侧是平滑和完全平行的。另外请注意, 肩部被切割以标记切割方向 (参见图 8)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56235/56235fig6.jpg"> 图 6.在试样表面进行氯丁橡胶处理.(a) 为了把标本卡盘放回原来的位置, 切片的恰克和卡盘持有者用胶带标记, 并在边界处切割。(b) 用巴斯德吸管将试样润湿, 使用氯丁橡胶溶液。(c) 三-狭缝网格, 用于拾取串行段。手柄弯曲到60度 (c, 右)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56235/56235fig7.jpg"> 图 7.切片和木扶手的塑料盖。塑料盖有助于防止超薄切片中的气流。木扶手是用来做微妙的工作, 如检索串行部分。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56235/56235fig8v2.jpg"> 图 8.设置截面厚度和获得适当厚度的截面。在连续段数达到20后, 截面厚度设置为 10 nm。由于切片不能切割10纳米厚的部分, 没有新的节出现, 和以前的切割部分成为分离的刀口。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig8v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56235/56235fig9.jpg"> 图 9.通过协议获得的超薄剖面的示例.请注意, 切割后的五组长度约为 1.8 mm 的连续段已经分离。在这种特殊情况下, 超薄部分的中心的颜色是黄色的由于存在的标本。部分的其他部分, 不包含样品和仅由树脂组成, 是银色的在颜色。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56235/56235fig10.jpg"> 图 10."节-保持回路"。(a) 顶部视图 (上半部分) 和底部视图 (照片的下部)。(b) 节保持回路用于保存第三组连续段。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56235/56235fig11.jpg"> 图 11.串行部分的检索。(a) "水表面抬高回路" (WSRL).该循环的手柄可以从轴上移除, 这是可移动的。通过转动螺钉顶部, 也可以上下移动循环。(b) WSRL 被磁铁固定在刀台上。(c) 更密切地看待 (b)。(d) 在检索过程中循环、水面、剖面和网格的图示 (侧面视图)。超薄剖面可以通过降低裸网格与超薄截面平行而精确地检索到狭缝网格。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig11large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/56235/56235fig12.jpg"> 图 12.将网格放置在 Formvar 胶片上.在铝架上的 Formvar 膜上, 放置截面的网格与一小滴水放在一起。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig12large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/56235/56235fig13.jpg"> 图 13.染色管16。一个凹槽0.6 毫米深是沿长轴使用刀片。网格按正确的顺序放置在凹槽中。管子的一端被切割 (箭头头) 以标记其方向。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig13large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/56235/56235fig14.jpg"> 图 14.将网格放置在标本架上。(a) 网格按正确的顺序放置在标本架上。(b) 然后用 "网格毒贩" 固定网格。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig14large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/56235/56235fig15.jpg"> 图 15.安装在狭缝网格上的串行部分。该图显示了安装在一个网格上的18到25节。每个狭缝中的数字表示从第一节开始的序列号。第一个节比其他部分更粗 (请参见图 8)。请注意, 这些截面连接在一起并具有相同的粗细。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig15large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 16" class="xfigimg" src="/files/ftp_upload/56235/56235fig16.jpg"> 图 16.Parakaryon myojinensis12 的串行部分.左下方的数字表示相对于第一节的序列。该图显示了 12 67 完整的部分。这个细胞被发现有一个大的核组成的裸 DNA 纤维, 一个单一的核膜, 和共生类似细菌, 但没有线粒体。因此, 这个有机体似乎是一个中间生命形式演变从原到核12。N, 核。转载自山口和女人, 2014。具有权限的14 。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig16large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 17" class="xfigimg" src="/files/ftp_upload/56235/56235fig17.jpg"> 图 17.大肠埃希菌9 的串行部分.左下方的数字表示相对于第一节的序列。该图显示了12完整的部分。发现该细胞含有21700核糖体9。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56235/56235fig17large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy at JoVE.com

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

本研究为在透射电子显微镜下获取无昂贵设备的微生物的连续超薄切片提供了可靠、简便的程序。

一种在透射电镜中获取微生物连续超薄切片的方法

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

a-method-for-obtaining-serial-ultrathin-sections-microorganisms

Nanyang Technological University

Research

JoVE Journal

Engineering

14.0K 次观看.  Chiba University. 本研究为在透射电子显微镜下获取无昂贵设备的微生物的连续超薄切片提供了可靠、简便的程序。

视频: A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.

在液体中使用液体池透射电子显微镜揭示材料的动态过程

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted ...

科研

工程学

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. The aggressive scaling of feature sizes, both on devices and interconnects, leads to serious challenges to ensure the required product reliability. Standard reliability tests and post-mortem failure analysis provide only limited information about the physics of failure mechanisms and degradation kinetics. Therefore it is necessary to develop new experimental approaches and procedures to study the TDDB failure mechanisms and degradation kinetics in particular. In this paper, an in situ experimental methodology in the transmission electron microscope (TEM) is demonstrated to investigate the TDDB degradation and failure mechanisms in Cu/ULK interconnect stacks. High quality imaging and chemical analysis are used to study the kinetic process. The in situ electrical test is integrated into the TEM to provide an elevated electrical field to the dielectrics. Electron tomography is utilized to characterize the directed Cu diffusion in the insulating dielectrics. This experimental procedure opens a possibility to study the failure mechanism in interconnect stacks of microelectronic products, and it could also be extended to other structures in active devices.

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. This paper demonstrates the procedure of an in situ TDDB experiment in the transmission electron microscope, which opens a possibility to study the failure mechanism in microelectronic products.

在透射电子显微镜原位时间相关的介质击穿:一种可能性，了解微电子器件的失效机理

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. ...

Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline.

This protocol describes the operation of a liquid flow specimen holder for scanning transmission electron microscopy of AuNPs in water, as used for the observation of nanoscale dynamic processes.

使用扫描透射电子显微镜研究纳米大小的物体的动态过程中的液体

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a ...

Characterization of Ultra-fine Grained and Nanocrystalline Materials Using Transmission Kikuchi Diffraction

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and nanocrystalline materials. The traditional techniques associated with scanning electron microscopy (SEM), such as electron backscatter diffraction (EBSD), do not possess the required spatial resolution due to the large interaction volume between the electrons from the beam and the atoms of the material. Transmission electron microscopy (TEM) has the required spatial resolution. However, due to a lack of automation in the analysis system, the rate of data acquisition is slow which limits the area of the specimen that can be characterized. This paper presents a new characterization technique, Transmission Kikuchi Diffraction (TKD), which enables the analysis of the microstructure of UFG and nanocrystalline materials using an SEM equipped with a standard EBSD system. The spatial resolution of this technique can reach 2 nm. This technique can be applied to a large range of materials that would be difficult to analyze using traditional EBSD. After presenting the experimental set up and describing the different steps necessary to realize a TKD analysis, examples of its use on metal alloys and minerals are shown to illustrate the resolution of the technique and its flexibility in term of material to be characterized.

This paper provides a detailed method to characterize the microstructure of ultra-fine grained and nanocrystalline materials using a scanning electron microscope equipped with a standard electron backscatter diffraction system. Metal alloys and minerals presenting refined microstructures are analyzed using this technique, showing the diversity of its possible applications.

超细晶和纳米晶材料的表征采用透射菊池衍射

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and ...

Fabrication of Magnetic Nanostructures on Silicon Nitride Membranes for Magnetic Vortex Studies Using Transmission Microscopy Techniques

Electron and x-ray magnetic microscopies allow for high-resolution magnetic imaging down to tens of nanometers. However, the samples need to be prepared on transparent membranes which are very fragile and difficult to manipulate. We present processes for the fabrication of samples with magnetic micro- and nanostructures with spin configurations forming magnetic vortices suitable for Lorentz transmission electron microscopy and magnetic transmission x-ray microscopy studies. The samples are prepared on silicon nitride membranes and the fabrication consists of a spin coating, UV and electron-beam lithography, the chemical development of the resist, and the evaporation of the magnetic material followed by a lift-off process forming the final magnetic structures. The samples for the Lorentz transmission electron microscopy consist of magnetic nanodiscs prepared in a single lithography step. The samples for the magnetic x-ray transmission microscopy are used for time-resolved magnetization dynamic experiments, and magnetic nanodiscs are placed on a waveguide which is used for the generation of repeatable magnetic field pulses by passing an electric current through the waveguide. The waveguide is created in an extra lithography step.

A protocol for the fabrication of magnetic micro- and nanostructures with spin configurations forming magnetic vortices suitable for transmission electron microscopy (TEM) and magnetic transmission x-ray microscopy (MTXM) studies is presented.

用透射显微技术制备氮化硅薄膜磁性纳米结构的研究

Electron and x-ray magnetic microscopies allow for high-resolution magnetic imaging down to tens of nanometers. However, the samples need to be prepared ...

Single-Digit Nanometer Electron-Beam Lithography with an Aberration-Corrected Scanning Transmission Electron Microscope

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by employing an aberration-corrected scanning transmission electron microscope as the exposure tool. Here, we present results of single-digit nanometer patterning of two widely used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. The method achieves sub-5 nanometer features in poly (methyl methacrylate) and sub-10 nanometer resolution in hydrogen silsesquioxane. High-fidelity transfer of these patterns into target materials of choice can be performed using metal lift-off, plasma etch, and resist infiltration with organometallics.

We use an aberration-corrected scanning transmission electron microscope to define single-digit nanometer patterns in two widely-used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. Resist patterns can be replicated in target materials of choice with single-digit nanometer fidelity using liftoff, plasma etching, and resist infiltration by organometallics.

用畸变校正扫描透射电镜进行单数字纳米电子束光刻

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by ...

Graphene-Assisted Quasi-van der Waals Epitaxy of AlN Film on Nano-Patterned Sapphire Substrate for Ultraviolet Light Emitting Diodes

This protocol demonstrates a method for graphene-assisted quick growth and coalescence of AlN on nano-pattened sapphire substrate (NPSS). Graphene layers are directly grown on NPSS using catalyst-free atmospheric-pressure chemical vapor deposition (APCVD). By applying nitrogen reactive ion etching (RIE) plasma treatment, defects are introduced into the graphene film to enhance chemical reactivity. During metal-organic chemical vapor deposition (MOCVD) growth of AlN, this N-plasma treated graphene buffer enables AlN quick growth, and coalescence on NPSS is confirmed by cross-sectional scanning electron microscopy (SEM). The high quality of AlN on graphene-NPSS is then evaluated by X-ray rocking curves (XRCs) with narrow (0002) and (10-12) full width at half-maximum (FWHM) as 267.2 arcsec and 503.4 arcsec, respectively. Compared to bare NPSS, AlN growth on graphene-NPSS shows significant reduction of residual stress from 0.87 GPa to 0.25 Gpa, based on Raman measurements. Followed by AlGaN multiple quantum wells (MQWS) growth on graphene-NPSS, AlGaN-based deep ultraviolet light-emitting-diodes (DUV LEDs) are fabricated. The fabricated DUV-LEDs also demonstrate obvious, enhanced luminescence performance. This work provides a new solution for the growth of high quality AlN and fabrication of high performance DUV-LEDs using a shorter process and less costs.

A protocol for graphene-assisted growth of high-quality AlN films on nano-patterned sapphire substrate is presented.

紫外发光二极管纳米图案蓝宝石基板的AlN薄膜的石墨烯辅助准范德瓦尔斯皮Taxy

This protocol demonstrates a method for graphene-assisted quick growth and coalescence of AlN on nano-pattened sapphire substrate (NPSS). Graphene layers ...

In Situ Transmission Electron Microscopy with Biasing and Fabrication of Asymmetric Crossbars Based on Mixed-Phased a-VOx

Resistive switching crossbar architecture is highly desired in the field of digital memories due to low cost and high-density benefits. Different materials show variability in resistive switching properties due to the intrinsic nature of the material used, leading to discrepancies in the field because of underlying operation mechanisms. This highlights a need for a reliable technique to understand mechanisms using nanostructural observations. This protocol explains a detailed process and methodology of in&#160;situ nanostructural analysis as a result of electrical biasing using transmission electron microscopy (TEM). It provides visual and reliable evidence of underlying nanostructural changes in real time memory operations. Also included is the methodology of fabrication and electrical characterizations for asymmetric crossbar structures incorporating amorphous vanadium oxide. The protocol explained here for vanadium oxide films can be easily extended to any other materials in a metal-dielectric-metal sandwiched structure. Resistive switching crossbars are predicted to serve the programmable logic and neuromorphic circuits for next-generation memory devices, given the understanding of the operation mechanisms. This protocol reveals the switching mechanism in a reliable, timely, and cost-effective way in any type of resistive switching materials, and thereby predicts the device&#39;s applicability.

In Situ Transmission Electron Microscopy with Biasing and Fabrication of Asymmetric Crossbars Based on Mixed-Phased a-VOx

Presented here is a protocol for analyzing nanostructural changes during in&#160;situ biasing with transmission electron microscopy (TEM) for a stacked metal-insulator-metal structure. It has significant applications in resistive switching crossbars for the next generation of programmable logic circuits and neuromimicking hardware, to reveal their underlying operation mechanisms and practical applicability.

基于混合相位a-VOx的不对称横杆的偏置和制造现场传输电子显微镜

Resistive switching crossbar architecture is highly desired in the field of digital memories due to low cost and high-density benefits. Different ...

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at length scales down to the atomic level. In situ closed-cell gas reaction (CCGR) studies performed using (scanning) transmission electron microscopy (STEM) can separate and identify localized dynamic reactions, which are extremely challenging to capture using other characterization techniques. For these experiments, we used a CCGR holder that utilizes microelectromechanical systems (MEMS)-based heating microchips (hereafter referred to as &#34;E-chips&#34;). The experimental protocol described here details the method for performing in situ gas reactions in dry and wet gases in an aberration-corrected STEM. This method finds relevance in many different materials systems, such as catalysis and high-temperature oxidation of structural materials at atmospheric pressure and in the presence of various gases with or without water vapor. Here, several sample preparation methods are described for various material form factors. During the reaction, mass spectra obtained with a residual gas analyzer (RGA) system with and without water vapor further validates gas exposure conditions during reactions. Integrating an RGA with an in situ CCGR-STEM system can, therefore, provide critical insight to correlate gas composition with the dynamic surface evolution of materials during reactions. In situ/operando studies using this approach allow&#160;for detailed investigation of the fundamental reaction mechanisms and kinetics that occur at specific environmental conditions (time, temperature, gas, pressure), in real-time, and at high spatial resolution.

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods.

在传输电子显微镜中执行 原位 闭电池气体反应

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at ...

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the surgical and instrumental expertise required to accurately quantify the structure and organization across multiple length scales have stunted its advancement. This work provides a detailed description of the murine dissection, en bloc staining, sample processing, and correlative imaging procedures for depicting the heart valve at different length scales. Hydrostatic transvalvular pressure was used to control the temporal heterogeneity by chemically fixing the heart valve conformation. Micro-computed tomography (&#181;CT) was used to confirm the geometry of the heart valve and provide a reference for the downstream sample processing needed for the serial block face scanning electron microscopy (SBF-SEM). High-resolution serial SEM images of the extracellular matrix (ECM) were taken and reconstructed to provide a local 3D representation of its organization. &#181;CT and SBF-SEM imaging methods were then correlated to overcome the spatial variation across the pulmonary valve. Though the work presented is exclusively on the pulmonary valve, this methodology could be adopted for describing the hierarchical organization in biological systems and is pivotal for the structural characterization across multiple length scales.

Here, we describe a correlative workflow for the excision, pressurization, fixation, and imaging of the murine pulmonary valve to determine the gross conformation and local extracellular matrix structures.

穆林肺瓣膜相关成像的手术和样品处理

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the ...