We thank the Pathology Research Core for assistance with electron microscope analysis studies at Cincinnati Children's Hospital Medical Center. The work was supported by NIH (American Society of Hematology Bridge award to-MDF; R01 DK102890 to MDF).

所有试验程序都通过在辛辛那提儿童研究基金会的体制动物委员会批准。 4个月 - 在本研究中，造血干细胞从C57BL / 6年龄2近交系小鼠的骨髓中分离。细胞使用具有不同表面标志包括世系，的c-Kit，的Sca-1的BM染色后的FACS分选，Flt3的和CD34用于基于Lin-的的Sca-1 +的c-Kit +的Flt3的HSC纯化- CD34 -门控策略作为标准协议8如前所述。 注意:一些剧毒化学品在此过程中使用。这些是吸入和皮肤接触有毒。请戴手套和防护服。在通风橱中使用这些化学品工作时的工作。 1.细胞，固定，染色和预嵌入<ol><li>排序万的造血干细胞（使用林-的Sca-1 <suP&gt; +的c-Kit +的Flt3 - CD34 -门控策略; LT-HSC种群）中含有1.5 ml离心管600微升的Iscove氏改良的Dulbecco氏培养基（IMDM）与使用FACS 10％胎牛血清。 </li><li>降速排序的造血干细胞在1000 XG使用的离心分离机摇出水桶在1.5 ml离心管10分钟。通过温和抽吸除去培养基，并在每个步骤中的管离开200微升培养基。在这个阶段，将细胞沉淀保留在管不可见的。 </li><li>通过加入0.2ml在室温（RT）下2个固定剂溶液1小时1,9固定细胞。 <ol><li>使用前固定液新鲜。在1倍，将溶液在0.1M二甲胂缓冲器包含3％多聚甲醛和2.5％戊二醛。 </li></ol></li><li>离心机使用具有开式水桶离心机将细胞在30℃1000×g离心10分钟。 </li><li>洗涤细胞用600微升0.1M的二甲胂缓冲采用离心法和离心后留下200微升缓冲在管。 </li><li>通过在二甲胂酸盐缓冲液中加入200微升的伊文思蓝的2毫克/毫升溶液染色固定的细胞和在室温下孵育20分钟。这些细胞会被蓝色。 </li><li>洗涤细胞3次用600微升的0.1M二甲胂酸盐缓冲液使用离心，每次离开200μl的缓冲液在管中。 </li><li>重悬在200μl缓冲液中的细胞和细胞悬浮液转移到0.5毫升管。离心机在1000×g离心用台式离心机10分钟。删除缓冲轻轻地，而不会干扰现在可见的微小细胞沉淀，并留下50微升的缓冲区管。 </li><li>琼脂糖加入200毫克低熔点到5ml的0.1M二甲胂缓冲剂在15ml试管为4％的琼脂糖溶液。通过在100毫升玻璃瓶中的管传送到沸水熔化琼脂糖和直至使用保持冷淡。 </li><li>加入200微升4％低熔点的琼脂糖溶于0.1M cacodylaTE缓冲液以在0.5ml管中的细胞悬浮液，并立即用台式离心机离心在1000 xg离心10分钟，在30℃。 </li><li>确认细胞沉淀在0.5毫升管的底部进入离心后半固体琼脂糖之后，立即转移管至4℃或冰上20分钟，以固化琼脂糖。这一步骤是非常关键的，因为在细胞的电子显微镜下的好簇此步骤的结果的小可见细胞团块。 </li><li>小心含有从管的细胞沉淀凝固琼脂糖转移到用27克针3,10含有缓冲器35毫米塑料培养皿中。 </li><li>修剪含细胞沉淀凝固琼脂糖成一体的约1 - 用手术刀2毫米。含细胞沉淀琼脂糖片转移至新的1.5毫升管。 </li><li>通过添加和使用吸管不离心除去缓冲用600微升0.1M的二甲胂缓冲液的3倍 - 2洗净。使用含有用于下一步骤将细胞沉淀此琼脂糖片。在该步骤中，样品可以移动到下一个步骤之前过夜储存在4℃。 </li></ol> 2. Osmification，脱水，嵌入<ol><li>从1.5 ml管与含有琼脂糖用吸管将细胞沉淀除去二甲胂酸盐缓冲液中。加1.0毫升1％四氧化锇（OSO 4）溶液在通风橱中，并孵育在4℃下1小时。 </li><li>用600微升的0.1M二甲胂缓冲液洗三次，将样品与帽转移到20ml的玻璃闪烁瓶中。 </li><li>处理用于脱水，浸润样品和嵌入到树脂（ 例如 ，LX-112），在6孔板以下解决方案的系列变化。移动使用镊子小区沉淀琼脂糖片。 <ol><li>沉浸在RT在25％的乙醇的样品15分钟。 </li><li>沉浸在RT在50％乙醇的样品15分钟。 </li><li>浸入样品中75％的乙醇在室温15分钟。 </li><li>沉浸在RT在95％乙醇的样品15分钟。 </li><li>沉浸在RT在100％乙醇的样品15分钟，两次。 </li><li>浸入样品中乙醇:树脂（3:1）在RT下30分钟。 </li><li>浸入样品中乙醇:树脂（1:1）在RT下30分钟。 </li><li>浸入样品中乙醇:树脂（1:3）在RT 30分钟。 </li><li>沉浸在RT在纯树脂试样60分钟，两次。 </li></ol></li><li>将样品转移至小心使用镊子金字塔尖带状橡胶模具的底部，并添加在样品的顶部更纯树脂填充模具。孵育样品在60℃下72小时对树脂聚合。 </li><li>通过扭转橡胶模具中取出从模具中聚合的树脂金字塔。细胞的黑色小簇应在聚合树脂金字塔可见，如前面7所述。附加聚合的树脂金字塔使用氰基丙烯酸酯安装筒。 </li></ol><ol><li>修剪用刀片和一个超薄切片机安装块之后金刚石刀金字塔块。使与顶部和块平行的彼此底部的短宽的梯形形状，并与均匀地成角度侧面。金字塔的这种形状有助于对连续切片。 </li><li>此外，修剪与周围的钻石刀细胞沉淀块，并删除周围的细胞沉淀琼脂糖片塑料部分。 </li><li>使用超薄切片机切1微米的部分。移动从水中船1微米的部分使用睫毛工具，如先前报道7的金属环的工具载玻片。在载玻片上并转让滑动到热板（37℃）干燥转印部。 </li><li>添加的甲苯胺蓝溶液来使用装有0.22微米过滤器，并孵育3分钟的注射器的部分下降。用蒸馏水冲洗和L的幻灯片等章节干燥。检查用光学显微镜，以确定细胞的位置。 </li><li>切割超薄切片 - 使用钻石刀（70 100纳米）。移动2 - 从供水船200目网格3段使用睫毛工具的玻璃培养皿。 </li><li>传送包含与部分网格玻璃培养皿的热板上在30℃下干燥段为30分钟。 </li><li>染色以1％乙酸双氧铀滴网格在室温10分钟，然后在蒸馏水中漂洗6 - 8倍。染色与雷诺柠檬酸铅，在室温下5分钟，在蒸馏水中冲洗6 - 8倍。传送包含网格来的热板上的玻璃培养皿，在37℃干燥的部分。 </li><li>用电子显微镜分析的部分中，在80千伏7。 </li></ol>

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作者什么都没有透露。

该方法能够在低细胞数目TEM分析，通过使用伊文思蓝染色和琼脂糖嵌入脱水，树脂包埋期间本地化一个微小的细胞沉淀和切片工序。重要的是，它保持细胞簇一起，并保留在细胞的超结构，这是可取的，以检查数据的量化的多个小区。 在TEM研究，含有数百万个细胞的细胞团，常常需要用于有效埋入到琼脂糖/明胶基质和用于洗涤，脱水和渗透到从大量细胞中获得的数据。研究人员已经在一个BSA的双丙烯酰胺聚合树脂通过的细胞悬浮液的固定化，在薄支撑单层，或用在低细胞数目/稀少样品4-6 TEM分析细胞离心涂片的染色。然而，它仍然是一个冗长乏味的工作，以定位和切割的细胞在TEM下，因为细胞的基质中的稀疏分布的和细胞形态的可能改变，由于扩散和细胞离心涂片制备过程中，往往需要再嵌入切片前的小区之间的裂解。此外，单层仍然具有挑战性本地化的切片11。因此，即使从低细胞数量的稀少生物样品细胞沉淀为TEM分析1容易得多。 此方法表示在从有限的样本的定性和定量的超结构分析方面以前发表的方法4,5-一个显著优势。一起，该协议描述了一种有效的方式来定位使用伊文思蓝，这从低细胞数量容易使样本处理用于TEM启用定性和定量数据分析用于TEM细胞沉淀。通常情况下，用于TEM的样品制备需要，因为在样品处理F中的多个步骤的细胞比通常所需的光学显微镜的数量较多，或TEM和用于在合适的位置1,10切割单元块的正确方向。 已知的是定影后osmification提供图像的更好的对比度，特别是那些膜和糖原颗粒的电子显微镜9中 ，并且还支承染色醋酸铀，用于蛋白质的非特异性相对染色下，和柠檬酸铅，即污迹膜，核酸和糖原。然而，osmification几乎是不可能的，在悬浮稀少生物样品中的BSA-BA预嵌入后执行由于样品损失的切片和矩阵变暗期间的高可能性。因此，电子显微镜的质量在以往的研究4,9被攻破。这里Osmification导致与在琼脂糖的颜色变化最小细胞沉淀变黑。因此，本协议允许osmification后嵌入预琼脂糖，以提供在TEM IM良好的对比度年龄。 细胞簇很容易用这种方法电镜分析。预染色伊文思蓝帮助定位样品制备过程中微小的细胞团，而不影响子细胞器单元的详细信息。此方法提供了样品制备成功地从低细胞数进行TEM分析的另一种方法。 （ - 8微米5）在该方法中，使用小尺寸的电池被有效地进行TEM分析。因此，这种方法可以很容易地与较大的细胞，如成纤维细胞和内皮细胞的甚至更低的细胞数目（〜1000个细胞）通过。虽然Evans蓝不影响本研究的子细胞器的细胞细节，使用伊文思蓝与低细胞数免疫金电子显微镜的未经过测试。 一起，描述了样品制备从低细胞数进行TEM研究的有效方法。需要TEM研究揭示critica在形态和细胞的超结构l信息。因此，超结构信息经常缺少对各种稀有细胞群。这种选择和样品制备新方法将有助于从任何罕见的细胞群进行TEM研究。此外，这种方法允许从细胞团的分析定性和定量的数据。作为电子显微镜提供细胞超结构和组织的无与伦比的细节，此方法将是非常有用的，以了解对关键许多生物过程，但罕见的，细胞群。

细胞的超微结构和亚细胞器的细节已通过透射电子显微镜（TEM）从组织或细胞沉淀1,2-研究中主要显露。组织坚固件可以很容易地用于电子显微镜研究。然而，TEM分析1,3悬浮细胞仍然充满挑战和必要的高细胞数， 即数百万个细胞。正因为如此，在悬浮稀有细胞群的超结构分析， 例如，造血干细胞（HSC），不容易评估。对于使用细胞离心涂片或单层方法稀少样品TEM分析多次尝试未能从细胞的数量有限得到显著定量数据。因此，高泡孔数的要求限制了使用这一强大的工具的理解稀有细胞群的亚细胞超微结构的信息。 为TEM研究的一个重要的限制与细胞数目有限S IN悬挂是细胞处理的定位和由此，从有限的细胞，特别是小尺寸的TEM研究仍然充满挑战。几种替代方法已被采用，以对付这种限制:BSA /双丙烯酰胺（BSA-BA）细胞悬浮液的介导聚合，将细胞与染料以使它们在薄膜支持，包括盖玻片可见的染色， 透射电镜和从细胞离心制剂4-6分析。然而，非常有限的成功达到了，因为很少有细胞在超切片后各部分找到。识别稀疏细胞的主要挑战仍然存在，因为单层细胞保持在固化的凝胶切片大多不可见的。此外，细胞的细胞离心涂片器制剂可以改变它们的细胞组织由于细胞扩散和细胞结构的脆弱性。因此，这些固有的缺陷认股权证以罕见的细胞群与更多的执行TEM研究的新方法一致性。为了克服这个问题，我们已经描述了一种新的和替代的样品制备方法用于从稀有细胞群7 TEM研究。 这里，我们报告的有效样品制备协议从稀少生物样品具有一致的定性和定量的结果执行的TEM。固定后进行伊文思蓝染色来本地化来自低数量的细胞， 即 10000骨髓造血干细胞和祖细胞，否则仍然可见一个微小的细胞沉淀，并将沉淀物嵌入琼脂糖之前脱水和树脂包埋过程。此方法聚类细胞一起，使超微结构和造血干细胞的亚细胞组织（标识为林-的Sca-1 +的c-Kit +的Flt3 - CD34 - ; HSC）的有效分析，难得0.2 - 0.5％的细胞群在骨髓中。这一实验亲母育可以进行超结构的研究，并取得许多罕见但非常重要的群体的定量结果非常有用。

<table><tbody><tr><td>Paraformaldehyde 20% Aqueous Soln</td><td>Electron microscopy Sciences</td><td>15713</td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic via inhalation, skin contact. Wear gloves, use fume hood.</td></tr><tr><td>Gluteraldehyde 70% Aqueous Soln</td><td>Electron microscopy Sciences</td><td>16350</td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic via inhalation, skin contact. Wear gloves, use fume hood.</td></tr><tr><td>Cacodyladate buffer&amp;nbsp;</td><td>Electron microscopy Sciences</td><td>12300</td><td>Make 0.1 M Cacodyladate buffer (pH 7.4) in distilled water, store at 4 &amp;deg;C. &lt;strong&gt;CAUTION&lt;/strong&gt; Irritant by inhalation or skin contact, wear gloves and work in fume hood.</td></tr><tr><td>Evans Blue&amp;nbsp;</td><td>Sigma</td><td>E-2129</td><td></td></tr><tr><td>Low melting agarose&amp;nbsp;</td><td>Sigma Aldrich</td><td>A-5030</td><td></td></tr><tr><td>Osmium tetraoxide</td><td>Electron microscopy Sciences</td><td>19130</td><td>1 g in 50 ml of 0.1 M cacodyladate buffer to make 2% OsO&lt;sub&gt;4&lt;/sub&gt; stock, &lt;strong&gt;CAUTION&lt;/strong&gt; Very toxic by inhalation or skin contact, strong oxidizer, wear protective clothing, eye protection, use fume hood.</td></tr><tr><td>&lt;strong&gt;LX-112 Embedding Kits (LADD&amp;reg;)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DDSA (Dodecenylsuccinic anhydride)</td><td>LADD</td><td>21340</td><td>Mix 16% DDSA; 30% NMA; 54% LX-112 together and then add 1.4% DMP-30</td></tr><tr><td>NMA (Nadic methyl anhydride)</td><td>LADD</td><td>21350</td><td></td></tr><tr><td>Epoxy Resins Epon 812 (LX-112)</td><td>LADD</td><td>21310</td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic by inhalation, skin contact. Wear gloves, protective clothing, use fume hood.</td></tr><tr><td>DMP-30 (2,4,6-Tri(dimethylaminomethyl)phenol)</td><td>LADD</td><td>21370</td><td></td></tr><tr><td>Reynolds lead citrate (EM Stain II)</td><td>Leica</td><td></td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic by inhalation, avoid contact with skin or eyes.</td></tr><tr><td>Uranyl acetate (EM Stain I)</td><td>Leica</td><td>8072820</td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic by inhalation, avoid contact with skin or eyes.</td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>ATCC</td><td>30-2005</td><td>composition: NaCl, NaHCO&lt;sub&gt;3&lt;/sub&gt;, HEPES, Glucose, Ca, Mg, sod pyruvate &lt;em&gt;etc.&lt;/em&gt; For details https://www.atcc.org/~/media/Attachments/E/9/7/E/4893.ashx</td></tr><tr><td>Pyramid tip mold&amp;nbsp;</td><td>Ted Pella</td><td>10585</td><td></td></tr><tr><td>mounting cylinders</td><td>Ted Pella</td><td>10580</td><td></td></tr><tr><td>Ultra-microtome&amp;nbsp;</td><td>(Leica EM UC7)</td><td></td><td></td></tr><tr><td>200 mesh grids (nikil)</td><td>Electron microscopy Sciences</td><td>G-200 Ni</td><td></td></tr><tr><td>Electron Microscope</td><td>Hitachi model H-7650&amp;nbsp;</td><td></td><td></td></tr><tr><td>Image capture engine software</td><td>AMT-600&amp;nbsp;</td><td></td><td></td></tr><tr><td>35 mm plastic Petri dishes</td><td>Fisher scientific</td><td></td><td></td></tr><tr><td>Quick Bond Super glue Cyanoacrylate&amp;nbsp;</td><td>Electron microscopy Sciences</td><td>72588</td><td></td></tr><tr><td>Razor blades</td><td></td><td></td><td></td></tr><tr><td>Glass scintillation vials&amp;nbsp;</td><td>Fisher scientific&amp;nbsp;</td><td>03-337-14</td><td></td></tr><tr><td>Glass slides&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Eyelash tool</td><td></td><td></td><td></td></tr><tr><td>Metal Loop tool&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

an alternative approach for sample preparation with low cell number for tem analysis

透射电子显微镜（TEM）提供了细胞组织和超微结构的细节。然而，稀有细胞群，尤其是细胞悬浮液，如造血干细胞（HSC）的TEM分析，仍然很有限，由于高细胞数的样品制备过程的要求。有用于从稀少样品TEM分析几个细胞离心涂片或单层的方法，但这些方法无法从细胞的数量有限得到显著定量数据。这里，对于在TEM研究的样品制备的替代的和新颖的方法是罕见的细胞群，使定量分析说明。 相对较低的细胞数量， 即10000造血干细胞，成功地应用于相比，数以百万计通常用于TEM研究细胞的TEM分析。特别是，多聚甲醛，戊二醛后进行伊文思蓝染色（PFA-GA）固定形象化狭小的牢房像素让，这有利于在琼脂糖嵌入。在超薄切片观察到大量细胞群。细胞有保存完好形貌，并高尔基复合体和几个线粒体的超结构细节也清晰可见。这种高效，容易和可重复的协议允许从低细胞数的样品制备和可用于对从有限的生物样品稀有细胞群的定性和定量TEM分析。

样品制备的高效和一致的协议来执行从描述的细胞数量低TEM分析。伊文思蓝和细胞转移到0.5 ml离心管后固定染色有助于可视化的微小细胞沉淀7。 Osmification在琼脂糖四氧化锇导致脱水和嵌入在一个简单的检测暗细胞沉淀。 从含有微小的细胞沉淀块半薄切片证实许多小区的集群一起（ 图1A和1B）。超薄切片从细胞聚集区切割并在电子显微镜下（ 图1C）进行分析。这些细胞的形态和超结构进行了很好地保存（ 图1D）。单个细胞的电子显微照片显示对细胞内高细节超结构（ 图1E）。此外，高倍率（50,000X）图像显示了保存完好的结构和亚细胞细胞器，诸如线粒体（ 图1F）的完整性。 <img alt="图1" src="/files/ftp_upload/54724/54724fig1.jpg" /> 图1: 图像在光镜和分析电子显微镜。 （A）的 1微米厚的光显微镜下甲苯胺蓝染色的部分的代表性领域的低放大率图像。（B）的光显微镜图像示出了簇细胞的1微米厚部分的甲苯胺蓝染色之后的完整的形态。 （℃）醋酸铀染色和柠檬酸铅后电子显微镜下观察到的小区集群的概述字段（D）的单个的LT-HSC电子显微镜下（E）高等MAGNIFHSC的ication图像显示的亚细胞结构。（F）从HSPC线粒体的高倍图像。比例尺:A / B，100微米; C，10微米; D，2微米; E / F，500纳米。 &#160; <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54724/54724fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis at JoVE.com

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

an-alternative-approach-for-sample-preparation-with-low-cell-number

Universidad San Sebastian

Research

JoVE Journal

Biology

13.6K 次观看.  Cincinnati Children’s Research Foundation. Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis. 

视频: An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Sample Preparation and Experimental Design for In Situ Multi-Beam Transmission Electron Microscopy Irradiation Experiments

There is a need to understand materials exposed to overlapping extreme environments such as high temperature, radiation, or mechanical stress. When these stressors are combined there may be synergistic effects that enable unique microstructural evolution mechanisms to activate. Understanding of these mechanisms is necessary for the input and refinement of predictive models and critical for engineering of next generation materials. The basic physics and underlying mechanisms require advanced tools to be investigated. The in situ ion irradiation transmission electron microscope (I&#179;TEM) is designed to explore these principles.
To quantitatively probe the complex dynamic interactions in materials, careful preparation of samples and consideration of experimental design is required. Particular handling or preparation of samples can easily introduce damage or features that obfuscate the measurements. There is no one correct way to prepare a sample; however, many mistakes can be made. The most common errors and things to consider are highlighted within. The I&#179;TEM has many adjustable variables and a large potential experimental space, therefore it is best to design experiments with a specific scientific question or questions in mind.
Experiments have been performed on large number of sample geometries, material classes, and with many irradiation conditions. The following are a subset of examples that demonstrate unique in situ capabilities utilizing the I3TEM. Au nanoparticles prepared by drop casting have been used to investigate the effects of single ion strikes. Au thin films have been used in studies on the effects of multibeam irradiation on microstructure evolution. Zr films have been exposed to irradiation and mechanical tension to examine creep. Ag nanopillars were subjected to simultaneous high temperature, mechanical compression, and ion irradiation to study irradiation induced creep as well. These results impact fields including: structural materials, nuclear energy, energy storage, catalysis, and microelectronics in space environments.

Sample preparation techniques are outlined with specific considerations for in situ ion irradiation TEM experiments. Ion species, energy, and fluence are discussed with methods for how to select and compute them. Finally, procedures for conducting an experiment are described and accompanied by the representative results.

There is a need to understand materials exposed to overlapping extreme environments such as high temperature, radiation, or mechanical stress. When these ...

科研

工程学

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers of multi-layered samples. This removal of layers prior to plunge freezing can aid in reducing sample thickness to a level suitable for cryo-EM data collection, improving sample flatness, and facilitating image processing. The peel-blot technique allows for the separation of multilamellar membranes into single layers, of layered 2D crystals into individual crystals, and of stacked, sheet-like structures of soluble proteins to likewise be separated into single layers. The high sample thickness of these types of samples frequently poses insurmountable problems for cryo-EM data collection and cryo-EM image processing, especially when the microscope stage must be tilted for data collection. Furthermore, grids of high concentrations of any of these samples can be prepared for efficient data collection since sample concentration prior to grid preparation can be increased and the peel-blot technique adjusted to result in a dense distribution of single-layered specimen.

The peel-blot technique is a cryo-EM grid preparation method that allows for the separation of multilayered and concentrated biological samples into single layers to reduce thickness, increase sample concentration, and facilitate image processing.

剥离印迹技术:一种冷冻电镜样品制备方法，用于从多层或浓缩生物样品中分离单层

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers ...

生物化学

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native conditions, suspended in vitreous ice. This technique allows for the high-resolution visualization of proteins and other biomolecules without the need for crystallization, offering significant insights into their function and mechanism. Recent advancements in single-particle analysis, coupled with improved computational data processing, have made cryo-EM an indispensable tool in modern structural biology. Despite its growing adoption, cryo-EM faces persistent challenges that can limit its effectiveness, particularly uneven particle distribution. This issue often leads to poor resolution and reduced accuracy in reconstructed protein structures. This article outlines a simple, practical approach to address this challenge, using the small heat-shock protein from Methanocaldococcus jannaschii (MjsHSP16.5) as an example. The method optimizes sample preparation to minimize preferential adsorption, ensuring more homogeneous particle distribution and higher-quality protein cryo-EM structures. This technique offers valuable guidance for researchers aiming to overcome similar challenges in structural studies.

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

优化低温电子显微镜的样品制备

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native ...

An Unbiased Approach of Sampling TEM Sections in Neuroscience

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative studies of the changes in densities and distributions of such features, an unbiased sampling protocol is vital for reliable results. Here, we present a workflow for the image acquisition of brain samples. The workflow allows systematic uniform random sampling within a defined brain region, and the images can be analyzed using a disector. This technique is much faster than extensive examination of serial sections but still presents a feasible approach to estimate the densities and distributions of ultrastructure features. Before embedding, stained vibratome sections were used as a reference to identify the brain region under investigation, which helped speed up the overall specimen preparation process. This approach was used for comparative studies investigating the effect of an enriched-housing environment on several ultrastructural parameters in the mouse brain. Based on the successful use of the workflow, we adapted it for the purpose of elemental analysis of brain samples. We optimized the protocol in terms of the time of user-interaction. Automating all the time-consuming steps by compiling a script for the open source software SerialEM helps the user to focus on the main work of acquiring the elemental maps. As in the original workflow, we paid attention to the unbiased sampling approach to guarantee reliable results.

We introduce a novel workflow for electron microscopy investigations of brain tissue. The method allows the user to examine neuronal features in an unbiased fashion. For elemental analysis, we also present a script that automatizes most of the workflow for randomized sampling.

神经科学中透射电镜切片采样的一种无偏方法

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative ...

神经科学

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

低温电子显微镜样品制备借助于聚焦离子束

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

生物学

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 ...

<meta charset="utf8"></head><body>TEM 视网膜类器官样品制备的优化方案

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

<meta charset="utf8"></head><body>作者聚焦:使用 TEM 分析成熟视网膜类器官的突触超微结构

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

制备用于透射电子显微镜的视网膜类器官样品

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

对于样品制备与TEM分析低细胞数量的另一种方法

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