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。

<table><tbody><tr><td>JB-4 Plus Embedding Kit</td><td>Polysciences Europe GmbH</td><td>18570-1</td><td>includes Benzoyl Peroxide, Plasticized (Catalyst) and Solution A+B</td></tr><tr><td>Polyethylene Molding Cup Trays, 6 x 8 x 5 mm hexagon (9 cavities)</td><td>Polysciences Europe GmbH</td><td>17177A-3</td><td></td></tr><tr><td>Polyethylene Molding Cup Trays, 13 x 19 x 5 mm (9 cavities)</td><td>Polysciences Europe GmbH</td><td>17177C-3</td><td></td></tr><tr><td>JB-4 Plastic Block Holders</td><td>Polysciences Europe GmbH</td><td>15899-50</td><td></td></tr><tr><td>Eosin</td><td>Waldeck GmbH &amp;amp; Co. KG, Division Chroma</td><td>1A-196</td><td></td></tr><tr><td>Microtec CUT 4060E</td><td></td><td></td><td>rotary microtome</td></tr><tr><td>Leica DM LM, fluorescence compound microscope</td><td>Leica Mikrosysteme Handelges.m.b.H</td><td></td><td></td></tr><tr><td>GFP filter set</td><td>Leica Mikrosysteme Handelges.m.b.H</td><td>11090937180000</td><td></td></tr><tr><td>Motorised cross table</td><td>Walter Uhl, technische Mikroskopie GmbH &amp;amp; CO. KG</td><td>KT5-LSMA</td><td></td></tr><tr><td>Digital video camera SPOT-FLEX</td><td>Visitron Systems GmbH.</td><td></td><td></td></tr><tr><td>precisExcite High-Power LED</td><td>Visitron Systems GmbH.</td><td></td><td>light source</td></tr><tr><td>VisiView 2.1.4</td><td>Visitron Systems GmbH.</td><td></td><td>Image capturing software</td></tr><tr><td>Hard metal knife (tungsten carbide), profile D</td><td>Leica Mikrosysteme Handelges.m.b.H</td><td></td><td></td></tr><tr><td>KL 2500 LCD</td><td>Schott AG</td><td></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

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

高分辨率光纤内窥镜为原位细胞成像

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

科研

生物工程

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) ...

发育生物学

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.&#160;

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

用于扫描电子显微镜的胚胎、蛋壳和真菌培养

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, ...

环境科学

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 ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

生物学

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

<meta charset="utf8"></head><body>使用体积电子显微镜在 3D 中可视化视网膜突触结构

Preparation of Retinal Samples for Volume Electron Microscopy

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level precision. Within the retina, various types of neurons establish synaptic connections in the inner and outer plexiform layers. While conventional EM techniques have yielded valuable insights into retinal subcellular organelles, their limitation lies in providing 2D image data, which can hinder accurate measurements. For instance, quantifying the size of three distinct synaptic vesicle pools, crucial for synaptic transmission, is challenging in 2D. Volume EM offers a solution by providing large-scale, high-resolution 3D data. It is worth noting that sample preparation is a critical step in Volume EM, significantly impacting image clarity and contrast. In this context, we outline a sample preparation protocol for the 3D reconstruction of photoreceptor axon terminals in the retina. This protocol includes three key steps: retina dissection and fixation, sample embedding processes, and selection of the area of interest.

<meta charset="utf8"></head><body>作者聚焦：使用体电子显微镜进行视网膜神经科学研究

This protocol describes the detailed steps for preparing retinal samples for volume electron microscopy, focusing on the structural features of retinal photoreceptor terminals.

用于体电子显微镜检查的视网膜样品的制备

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level ...

神经科学

<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 ...

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

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高分辨率光纤内窥镜为原位细胞成像

使用3D图像的快速采集高分辨率显微镜落射

用于扫描电子显微镜的胚胎、蛋壳和真菌培养

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

采用先进的3D荧光显微镜定量分析自噬

内质网子域的培养细胞中的可视化

用于体电子显微镜检查的视网膜样品的制备

用于体电子显微镜检查的视网膜样品的制备

用于体电子显微镜检查的视网膜样品的制备

制备用于透射电子显微镜的视网膜类器官样品