作者感谢Tim Mohun在开发HREM和Petra Heffeter提供样品方面的无价贡献。

所有程序均按照维也纳医科大学的道德准则进行。 样品制备<ol><li> 准备胚胎和胚胎组织（高达5 x 5 x 5 mm 3 ） <ol><li>在4％PFA / PBS或Bouin固定剂中将胚胎或部分胚胎在4℃下固定至少16-24小时。 注意:可以使用几种物种和各种发育阶段的胚胎，例如48小时斑马鱼，19小时鸡和鹌鹑以及在这里使用的产前小鼠胚胎（见代表性结果 ）。收集标本并在室温下转移到PBS中进行分级，然后放入固定剂中。如果需要，在固定前用微型剪刀或手术刀修剪标本，以装入嵌入模具中。 </li><li>取出固定剂，并在PBS中以4℃恒温摇匀洗涤胚胎组织g进行24小时（2-3次更换）。 </li><li>将样品在70℃，80％，90％，96％乙醇中4℃脱水2〜3小时。将含有样品的管放在旋转器上。 注意:对于小样品（&lt;2 x 2 x 2 mm 3 ），样品搁置在每个乙醇中的时间可能会减少到60分钟。对于大样品（&gt; 5 x 5 x 5 mm 3 ），可能会延长至4 h。 </li><li>在穿防护手套的通风橱下执行以下渗透步骤。 <ol><li>通过向100mL溶液A中加入1.25g过氧化苯甲酰（增塑催化剂）和0.4g曙红来制备渗透溶液（更多细节参见材料表 ）。在4℃的磁力搅拌板的烧杯中搅拌，直到曙红和催化剂完全溶解（2-3小时）。 </li><li>将样品置于浸泡溶液中12-24小时。在连续摇摆或旋转下保持在4°C。 3和12之后用新鲜溶液代替浸润溶液H。 注意:对于大胚胎（&gt; 1厘米冠/臀部长度），将浸润时间延长至48小时。 </li></ol></li></ol></li><li> 准备成人组织样品（高达5 x 5 x 7 mm 3 ） <ol><li>在4℃下将含有4％碳酸的2％PFA / PBS中的组织固定至少3天。 注意:这种溶液可以固定在几种新生儿和成年人，啮齿动物，猪，果蝇和斑马鱼的皮肤，肝脏，胰腺，肾脏，甲状腺，心脏，横纹肌，脑，神经和肿瘤模型中产生的组织样本然后进行HREM成像处理（我们展示样品的固定;见代表性结果 ）。用解剖刀，剪刀或活检冲孔冲洗组织样品，并将其直接转移到固定剂中。用于固定神经组织使用4％PFA / PBS。 </li><li>取下固定剂，并在自来水下清洗组织3-6小时。 </li><li>通过随后的plac去除样品在70％，80％，90％，96％乙醇中混合0.4g曙红/ 100mL（每次2-3小时）。将样品保持在4°C的恒定摇摆或旋转器下。 注意:对于小样品（&lt;2 x 2 x 2 mm 3 ），样品搁置在每个乙醇中的时间可能会减少到60分钟。对于大样品（&gt; 5 x 5 x 5 mm 3 ），可能会延长至4-8 h。 </li><li>在穿防护手套的通风橱下执行以下渗透步骤。 <ol><li>通过向100mL溶液A中加入1.25g过氧化苯甲酰（增塑催化剂）和0.4g曙红来制备渗透溶液（更多细节参见材料表 ）。在4℃的磁力搅拌板的烧杯中搅拌，直到曙红和催化剂完全溶解（2-3小时）。 </li><li>将样品置于浸泡溶液中24 - 36 h。在连续摇摆或旋转下保持在4°C。 3和3之后用新鲜溶液代替浸润溶液12小时。 注意:对于大样品（&gt; 5 x 5 x 5 mm 3 ），渗透时间延长至48 h。 </li></ol></li></ol></li><li> 准备其他有机材料 注意:用普通剪刀将皮肤替代物和纸张修剪成所需的尺寸。不需要固定。 <ol><li>跳过固定，洗涤和脱水步骤。 </li><li>在穿防护手套的通风橱下执行以下渗透步骤。 <ol><li>通过向100mL溶液A中加入1.25g过氧化苯甲酰（增塑催化剂）和0.4g曙红来制备渗透溶液（更多细节参见材料表 ）。在4℃的烧杯中，在磁力搅拌板上搅拌直到曙红和催化剂完全溶解（2-3小时）。 </li><li>将样品置于浸泡溶液中3 - 12 h。在连续摇摆或旋转下保持在4°C。 1和2 - 4 h后用新鲜溶液代替浸润溶液。 </li></ol> &lt;/ LI&gt; </ol></li></ol>嵌入注意:请在通风柜下方戴上防护手套的所有步骤。 <ol><li>如上所述准备新鲜的渗透溶液（步骤1.1.4.1）。加入4 mL溶液B（详见表格材料 ）至100 mL渗透溶液中，制备嵌入溶液。 注意:如果需要在嵌入模具中确定样品的精确取向，并允许平行地嵌入几个样品，则可以将烧杯放入冰浴中减慢储液的聚合。 </li><li>使用常规体积为6 x 8 x 5 mm 3或13 x 19 x 5 mm 3的模具，或具有高达8 x 10 x 15 mm体积的定制模具，使用成型杯托盘（详见表格 ） 3 （ 图2 ）。 </li><li>用模具填充模具，并将样品转移到模具中勺子。在传输过程中，确保样品完全被包埋溶液覆盖，以避免空气捕获。 注意:这可以在室温下进行，如果从冰箱收集样品和嵌入溶液后立即开始。 <ol><li>使用针或镊子将样品定位在模具内。 </li><li>一旦嵌入溶液开始变粘，将模具夹（ 见表材料 ）放在模具的顶部，并通过块支架的中心孔添加嵌入溶液，直到块支架的底部覆盖1-2毫米的嵌入溶液。 </li><li>通过用液体石蜡覆盖成型杯托盘，并等待它变硬。或者将第二个成型杯托盘倒置在带有样品的成型杯托盘上，并用胶带将其密封以防风。 </li></ol></li><li>通过将密封的模制杯托盘存放1 -室温2天。 </li><li>对于后聚合加工，将具有聚合块的成型杯托盘放置在标准实验室烘箱中，并在70-80℃下烘烤至少1-2天，直到变成深红色。从烤箱收集托盘，并从模具中取出块体。 注意:烘烤可以在正常的环境条件下进行。覆盖成型杯托盘，可以确保在嵌入后的第一相中的密封密封，以便在10小时后检查树脂的颜色。在烘烤后立即，树脂柔软，并且块可以容易地从模杯托盘移除。在2-3小时之内，它们很硬，可以在室温下储存几个月。 </li></ol> 3.数据生成注意:此处使用的HREM原型18,25包括以下项目:（i）具有块支架的旋转切片机在其上部转折点的每次切割后停止。 （ii）标准，非一次性切片刀，硬质合金，型材D（详见表格 ）。 （iii）具有目标左轮手枪的荧光复合显微镜头和其光轴上的GFP滤光镜立方体（激发470/40;发射滤光片525/50）。光轴垂直于安装在切片机上的块的新鲜切割表面设置，并且由允许上下移动的装置保持。 （iv）携带切片机的电动十字台。工作台可沿光轴方向移动，横向移动。 （v）连接到荧光复合显微镜的数码摄像机。 （vi）单色光源（470nm）。 （vii）与数码相机连接的计算机，具有数据生成软件（详见表格 ）。 <ol><li> 程序 <ol><li>指示感兴趣的区域<ol><li>使用标准实验室（参见表格 ），用于将白光倾斜地导向块表面。 </li><li>当嵌入材料投射到块表面时，确定嵌入材料的轮廓。使用笔或刀片在块体表面上指示感兴趣区域和后续视场。 注意:此步骤必须在分割之前定义视野范围。 </li></ol></li><li>定义视野。 <ol><li>将树脂块用样品安装在切片机上。 </li><li>将挡块移动到其停止位置（挡块保持架偏移的上转弯点）。 </li><li>启动数码相机和数据生成软件，并获取实时图像。 </li><li>选择一个物镜，以适当的放大倍率来覆盖块表面上指示的感兴趣区域。 注意:通常使用2.5X，5X，10X，20X放大倍数和0.07和0.40之间的数值孔径的目标。 </li&gt; <li>使用电动交叉表横向移动光学器件和切片机，直到感兴趣的区域与计算机屏幕上显示的视野匹配。 </li></ol></li><li>焦点。 <ol><li>执行一步一步的手动分段，直到样品的第一个结构变得可见。 </li><li>通过沿着光轴的方向移动切片机，在光学元件的聚焦平面中排列块表面。 </li><li>选择部分厚度;推荐厚度为0.5至5μm的部分。 </li></ol></li></ol></li><li> 数据生成和处理 <ol><li>开始编排分段和图像捕获的软件程序。遵循成像捕获软件的文档说明。 </li><li>一旦样品完全分割，就停止软件程序并将一个缩放滑块放在块表面的前面。 </li><li>以交互方式捕获图像以供以后校准灰。 注意:必须在新安排的每一次使用目标时进行。 </li><li>将图像系列转换为.jpg格式的8位灰度图像。 </li><li>将图像系列加载到3D可视化软件中并调整对比度。 注意:按照相应软件的说明，图像转换和3D可视化可以使用各种商业或免费的标准软件包完成。 </li></ol></li></ol>

作者没有什么可以披露的。

HREM是一种高度可靠的微观方法，非常适用于生物医药和工业中使用的广泛有机材料的可视化18,21,26,27,28,29,30,31,32,33,34,35,36,37 ， 38,39,40 。它可以用作独家成像模式，如目前发展障碍机制（DMDD）计划41，42 <sup&gt; 43,44 ，或作为多模式成像管线45的综合部分。 完整功能的HREM数据生成装置可以由常规实验室部件组装，并且包括电动切片机，显微镜，电动交叉表和具有适当软件的计算机25 。使用配备有块保持器的切片机至关重要，该切片机在定义的位置的每个部分和光学通路中的GFP滤光片之间可重复地停止。然而，全面运作的全包解决方案可以从Indigo Scientific等公司购买。 HREM面临与所有组织学技术相同的限制，除了在切片或切片安装期间不引入人为因素。然而，存在限制，这是由于在切片之前必须染色标本而产生的从嵌入材料的特点。通过整个样品渗透曙红需要获得足够的组织对比度;非常致密的材料，脂肪组织和无机物质有效阻碍曙红渗透，并导致物体中心的未染色组织。使用特殊的固定剂有助于染色皮肤标本，但仍然没有适当的方法来彻底克服这个问题。另一个限制是，在切片过程中阻挡高于2厘米的树脂会破裂。这可以通过单独切割样品和加工部件来部分避免。 在嵌入过程中，将小样本或具有不规则表面的样品正确定位在模具中通常是有问题的。用琼脂糖覆盖样品并按照方案进行处理琼脂糖块通常可以解决这个问题19 。一种替代方法，这也有助于块在组织中断要将其已经硬化的块从其固定器中移除，并按照所述的嵌入程序重新嵌入。 典型的HREM数据集包括500到3000个单个图像。其数值分辨率由连续图像之间的距离（ 即 ，通过截面厚度），相机目标的特性和所使用的光学器件的性质决定。我们使用1μm和5μm之间的截面厚度，并取得了很好的效果，尽管提出的协议并不完全消除伪影20,46的光泽。这些伪像是由位于块内深处的强烈染色的组织引起的，导致块表面上的组织信息模糊。 相机的目标尺寸为2,560 x 1,920像素2，2,048 x 2,048像素2和4,096 x 4,096像素2 ，并组合内置1.25X，2.5X，5X，10X和20X物镜。这导致0.18×0.18μm2和5.92×5.92μm2之间的数字像素尺寸，这被证明足以用于组织结构和细胞形状的3D分析，甚至可视化核。鉴于高数字分辨率，其他细胞器也应该可见。由于简单的伊红染色，造成的对比度不足，并且目标的光学性质大大降低了辨别结构的可能性。考虑到数值孔径的HREM数据的最大真实空间分辨率约为1×1×1μm3，因此仅允许有效区分大于3×3×3μm3的结构。 所有数字成像技术的一个常见问题是视野的尺寸之间的折衷，其定义了可以显示的样本的一部分d在相机目标上，以及图像的数字分辨率。视场越大，最大可能的数字分辨率越低46 。这里使用的HREM设置允许生成HREM数据，其视野范围为0.74 x 0.74 mm 2 （20X物镜），数字分辨率为0.18 x 0.18μm2和12.12 x 12.12 mm 2 （1.25X物镜），显示在2.96 x 2.96μm2的数字分辨率。另外，商业化的设置可以提供更大的观点，但以真正的解决为代价。然而，从DMDD程序47的主页上显示的数据显而易见，它们提供了出色的结果。

有机和无机材料的结构分析是理解其物理性能和功能的第一步。这种分析的基础通常是通过仔细观察组织切片获得的二维（2D）信息，具有提取组织结构细胞，细胞形态和拓扑，分子组成和生物力学性质的各种简单和复杂的成像方法1 ， 2，3 。然而，2D信息不适合研究空间复杂的布置。因此，在过去几十年中建立了越来越多的允许产生数字体积数据的体内和体外方法，并且还在开发中。 大多数卷数据生成方法的有条理的原则是生成虚拟堆栈数字图像显示通过对象的虚拟或物理分段获得的部分。如果部分图像正确对齐，则会创建一个卷，该卷可以在虚拟剖面中重新划分，或用于创建3D曲面和体绘制模型。用于可视化人类和较大生物样本的流行技术是磁共振断层扫描（MRT），计算机断层扫描（CT），正电子发射断层扫描（PET）和单光子发射计算机断层扫描（SPECT）。小样本通常使用微磁共振成像（μMRI），光学投影断层扫描（OPT），光学相干断层扫描（OCT），光声层析成像（PAT），基于组织切片的方法，共聚焦显微镜和电子断层扫描显示5,6 ， 7,8,9,10 <s， 11，12，13，14，15，16，17 。 产生小标本和组织学样本的数字数据的相对较新的体积数据生成技术是与Tim Mohun 18,19密切合作开发的HREM方法。它是一种简单的基于显微镜的技术，它从在切片机上切片的树脂嵌入材料产生数字体积数据。数据有助于细胞分析和组织结构细胞分布以及中等光学微观层面小特征的度量分析。 HREM生成堆叠的固有对齐的数字图像，似乎从e获取osin染色组织切片。组织对比度和数据分辨率相对于视野超过了用μCT，μMRI和OPT产生的数据，但是低于用共聚焦，光片和电子显微镜可获得的数据。然而，与后者相反，HREM能够使组织学质量中具有高达5×5×7mm 3的相对大体积的样品可视化。一些最近的研究提供了单一成像技术的优点和缺点的详细描述和比较，为了客观性，我们参考有关其局限性和潜在应用领域4,21,22,23 ， 24 。 本研究重点介绍HREM成像方法，旨在提供用于生成广谱有机材料的HREM数据的非常简单的方案，以及它们的应用实例。创建HREM数据的工作流程很简单，适用于可嵌入甲基丙烯酸酯树脂的所有材料（ 图1 ）。然而，样品制备中存在组织特异性差异，需要考虑。因此，我们提供了三种准备各种样品的标准方案。所有嵌入和数据生成协议步骤都是相同的。

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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">VisiView 2.1.4</td>
			<td style="width:115px;">Visitron Systems GmbH.</td>
			<td style="width:161px;"></td>
			<td>Image capturing software</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:231px;">Hard metal knife (tungsten carbide), profile D</td>
			<td style="width:115px;">Leica Mikrosysteme Handelges.m.b.H</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">KL 2500 LCD</td>
			<td style="width:115px;">Schott AG</td>
			<td style="width:161px;"></td>
			<td>light source </td>
		</tr>
	</tbody>
</table>

high-resolution episcopic microscopy (hrem) - simple and robust protocols for processing and visualizing organic materials

我们提供使用高分辨率显微镜（HREM）方法生成数字体积数据的简单协议。 HREM能够在1 x 1 x 1和5 x 5 x 5μm3之间的典型数字分辨率下对体积高达5 x 5 x 7 mm 3的有机材料进行成像。将样品嵌入甲基丙烯酸酯树脂中并切片在切片机上。在每个部分之后，使用位于连接到复合显微镜头的光电管上的数字摄像机捕获块表面的图像。光轴穿过绿色荧光蛋白（GFP）过滤器立方体，并与每个部分后方的保持架臂静止的位置对齐。以这种方式，产生一系列固有对准的数字图像，显示后续的块表面。在三维（3D）可视化软件中加载这样的图像系列有助于立即转换为允许虚拟的数字卷数据在各种正交和倾斜平面中进行激励，并创建体积和表面渲染的计算机模型。我们提出三种简单的组织特异性方案，用于处理各种有机标本，包括小鼠，小鸡，鹌鹑，青蛙和斑马鱼胚胎，人体活检材料，未涂布纸和皮肤替代材料。

HREM生成一系列固有排列的数字图像，与苏木精/伊红染色组织切片的图像类似。与2D截面图像相反，HREM图像的堆叠允许对3D中各种有机材料的组织结构，形态和拓扑的可视化和分析。高对比度通常有助于快速简单的3D可视化体积渲染计算机模型和半自动轮廓发现，用于生成表面渲染模型。 HREM数据的大小根据摄像机目标的大小，图像捕获的模式（8,12,16位，灰度级，颜色）和单块面部图像的数量而有所不同。 2,048像素×2,048像素的1,000个8位.jpg灰度图像的较小数据集的大小约为900 MB。 3,000个8位的大型数据集，灰色图像为4,096 pixel x 4,096像素的容量约为20 GB。 所提供的方案简单而稳健，在过去十年中，它们用于生成许多不同标本的HREM数据。协议第1.1节被优化用于处理长度达1厘米的生物医学模型生物的全胚胎，尺寸高达5×5×5毫米3的胚胎组织样品;我们使用这种方法来产生以下物种的胚胎的HREM数据:小鼠（ Mus musculus） ，小鸡（ Gallus gallus ），斑马鱼（ Danio rerio ），鹌鹑（ Coturnix coturnix） ，非洲爪蛙（ Xenopus laevis ），马（ 马尾草 ），马鞭草（ Oncopeltus fasciatus ），鳄鱼（ Crocodylia ）和章鱼（ 章鱼 ）。所有种类的数据质量优良（ 如图3 ） 3的青少年和成人组织样本，并用于可视化组织样本皮肤，肝脏，胰腺，肾脏，甲状腺，心脏，横纹肌，脑，神经和从人（ 智人 ），小鼠（ Mus musculus ），大鼠（ Rattus norvegicus ），猪（ Sus scrofa domestica ） furo ），果蝇（ 果蝇 ）和斑马鱼（ Danio rerio ）。虽然大多数标本（ 例如 图4 ， 动画1 ）的结果非常好，但皮肤（表皮）和大脑3×3×3毫米3的大脑标本的中心部分通常由于曙红穿透不足而保持未染色这些组织。 协议针对加工纤维有机材料进行了优化，用于可视化涂层纸，未涂布纸，天然皮肤替代材料和干细胞种皮真皮替代材料的纤维结构。这些样品易于处理。未涂布纸和大多数皮肤替代品的数据质量好（ 图5 ， 动画2 ）。无机材料阻碍曙红渗透时，涂布纸的加工出现问题。当加工皮肤替代品时，由于它们被渗透溶液部分消化，所以出现了另一个问题。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/56071/56071fig1.jpg" /> 图1:工作流程。红色框中显示的步骤需要根据样品特性进行修改。绿色框中的步骤就是那些在所有样品中都是相似的。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56071/56071fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/56071/56071fig2.jpg" /> 图2:定制模具。模具可以通过将原始模具中的孔切入，插入巴斯德吸管的灯泡并用造型材料进行密封。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56071/56071fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/56071/56071fig3.jpg" /> 图3:示例性可视化胚胎。 （A，B）胚胎期收获的小鼠胚胎，E = 9.5。 HREM剖面图如（A） </STRONG&gt;。显示表面的体积渲染3D模型如（B）所示。 （C）通过E15.5小鼠胚胎的颈部的体积渲染模型的虚拟矢状切片。 （D）发育汉堡汉密尔顿（HH）阶段的雏鸡胚胎18.心脑血管成分的腔的表面模型与所有胚胎组织的体积渲染相结合。刻度棒=200μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56071/56071fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/56071/56071fig4.jpg" /> 图4:示例性可视化成人组织样品。 （A）通过人类神经的HREM部分图像的一部分。 Inlay更详细地显示了图像的一部分。 （B）通过猪肝的HREM部分图像的一部分。注意nuclei。 （C）人体淋巴组织的HREM部分图像的一部分。 （D，E）人类拇指垫厚皮肤。体积渲染3D模型的整个活组织检查。 （D）通过HREM数据（E）在虚拟切除前方的动脉，静脉和神经的表面渲染模型。 （F）成年小鼠组织中的实验性肿瘤。通过HREM数据在三个虚拟部分前面渲染的体积渲染的3D模型。注意坏死部位（箭头）。刻度棒=200μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56071/56071fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/56071/56071fig5.jpg" /> 图5:示例性可视化纤维材料。 （A，B）无涂层，棕色纸张的HREM剖面图像。 （B） </strong&gt;显示（A）的一部分 。注意纤维及其内腔。 （C）涂层纸的HREM部分。请注意，纤维保持未染色。 （D）天然皮肤替代材料的体积渲染模型。注意纤维的不同口径和形式。刻度棒=100μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56071/56071fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="电影1" src="/files/ftp_upload/56071/56071mov1.jpg" /> 动画1:人体拇指贴的厚皮肤的体积渲染模型。组织块的大小约为4.2×2.7×2.7mm 3 。体素大小为1.07×1.07×2μm3。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56071/animation1.mp4" target="_blank">请点击此处查看此视频。 （右键单击下载。）</a> <pclass ="jove_content"fo:keep-together.within-page ="1"&gt; <img alt="电影2" src="/files/ftp_upload/56071/56071mov2.jpg" /> 动画2:原生皮肤替代材料的体积渲染模型。样品的大小约为1.2×0.85×0.4mm 3 。体素大小为0.54×0.54×2μm3。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56071/animation2.mp4" target="_blank">请点击此处查看此视频。 （右键单击下载。）</a>

Watch this Scientific Journal Video about High-resolution Episcopic Microscopy HREM - Simple and Robust Protocols for Processing and Visualizing Organic Materials at JoVE.com

High-resolution Episcopic Microscopy HREM - Simple and Robust Protocols for Processing and Visualizing Organic Materials

我们提供简单而强大的方法来处理各种物种的活检材料，生物医学模型生物的胚胎和其他有机组织的样品，以便通过高分辨率显微镜显微镜方法生成数字体积数据。

高分辨率显微镜（HREM） - 用于处理和可视化有机材料的简单和可靠的协议

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging ...

high-resolution-episcopic-microscopy-hrem-simple-robust-protocols-for

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials

10.1K 次观看.  Medical University of Vienna. 我们提供简单而强大的方法来处理各种物种的活检材料，生物医学模型生物的胚胎和其他有机组织的样品，以便通过高分辨率显微镜显微镜方法生成数字体积数据。 

视频: High-resolution Episcopic Microscopy HREM - Simple and Robust Protocols for Processing and Visualizing Organic Materials

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

科研

发育生物学

Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy

High-resolution episcopic microscopic (HREM) technology enables rapid acquisition of high-resolution digital volumetric and three-dimensional (3D) morphometric data. Here, we describe the detailed protocol to image the entire mouse embryo. The protocol consists of four major sections: sample preparation, embedding, image acquisition and finally, 3D visualization. The technology requires specimens to be stained with a fluorescent dye, which can be problematic for large or dense specimens. To overcome this limitation, we have improved the existing protocol to enhance tissue penetration of the dye by pretreating the specimen with a solution containing urea and sodium dodecyl sulfate. The protocol uses only routine laboratory equipment and reagents for easy adaptation in standard laboratory settings. We show that the resulting high-resolution 3D images faithfully recapitulate the detailed morphologic features of the internal organs of mouse embryos, thereby permitting morphometric analyses. Together, we present a detailed and improved protocol using standard laboratory equipment to acquire high-resolution 3D images of small and large sized specimens.

We describe a detailed protocol using high-resolution episcopic microscopy to acquire three-dimensional (3D) images of mouse embryos. This improved protocol utilizes a modified tissue preparation method to enhance penetration of the fluorescent dye, thereby permitting morphometric analysis of both small and large-sized specimens.

使用3D图像的快速采集高分辨率显微镜落射

High-resolution episcopic microscopic (HREM) technology enables rapid acquisition of high-resolution digital volumetric and three-dimensional (3D) ...

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

长期共焦成像根和下胚轴发育的简单室

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

协议的可视化类固醇机关及其互动器官在果蝇免疫染色<em&gt;果蝇</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

用于在大容量中查找特定细胞目标的串行剖面的多模式分层成像

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin adhesives. Two different poly-(dimethyl siloxanes) (PDMS and soft skin adhesive (SSA)), have been used for in depth investigation of biological effects and adhesive characteristics. The composite films consist of a flexible backing layer and an adhesive top coating. Both layers have been manufactured by doctor blade application technique. In the present investigation, the adhesive behavior of the composite films has been investigated as a function of the layer thickness or a variation of the Young&#39;s modulus of the top layer. The Young&#39;s modulus of PDMS has been changed by varying the base to crosslinker mixing ratio. In addition, the thickness of SSA films has been varied from approx. 16 &#181;m to approx. 320 &#181;m. Scanning electron microscopy (SEM) and optical microscopy have been used for thickness measurements. The adhesive properties of elastomer films depend strongly on the film thickness, the Young&#39;s modulus of the polymers and surface characteristics. Therefore, normal adhesion of these films on glass substrates exhibiting smooth and rough surfaces has been investigated. Pull-off stress and work of separation are dependent on the mixing ratio of silicone elastomers.
Additionally, the thickness of the soft skin adhesive placed on top of a supportive backing layer has been varied in order to produce patches for skin applications. Cytotoxicity, proliferation and cellular adhesion of L929 murine fibroblasts on PDMS films (mixing ratio 10:1) and SSA films (mixing ratio 50:50) have been conducted. We have shown here, for the first time, the side by side comparison of thin composite films manufactured of both polymers and present the investigation of their biological- and adhesive properties.

A protocol for the manufacturing process of polymeric thin film composite structures possessing either different Young&#39;s moduli or thicknesses is presented. Films are produced for advanced cell culture studies or as skin adhesives.

用于细胞培养和皮肤应用的薄膜复合硅弹性体: 制造和表征

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...