We thank Drs. Yichen Huang, Chunming Guo and Zhenfang Zhou for their technical support. This research was funded by NIH/NIDDK (1R01DK091645-01A1, XL) and American Heart Association (AHA, 13GRNT16950006, XL).

所有的动物用途被机构动物护理和使用委员会在波士顿儿童医院的批准。 1.样品制备<ol><li> 定时交配 <ol><li>在同一个笼子里放置一个C57BL6野生型男性和女性在一起。 </li><li>检查第二天一早形成了交配塞。交配塞（ 又名 ，阴道或交配插头）是硬化胶质沉积，阻止阴道。 </li><li>设置插件日期作为胚胎天0.5（E0.5）。 </li></ol></li><li> 小鼠胚胎的隔离 <ol><li>通过CO 2窒息安乐死怀孕女性。 </li><li>把小鼠仰卧上的吸收垫，并用70％乙醇喷雾腹部区域。 </li><li>提起皮肤，并用在手术剪尾腹部开始5毫米的切口。剪切和去除腹部皮肤充分暴露内脏</li><li>附近的弗吉尼亚州切吉娜切除整个子宫。 </li><li>沿角子宫着床点之间切割。每个段或孕应该有内部只有一个胚胎。 </li><li>保持在1×冰冷的磷酸盐缓冲盐水（PBS）子宫段。 </li></ol></li><li> 胚胎解剖 <ol><li>将每个孕在冰冷的PBS立视下一个单独的菜。 </li><li>取出子宫组织，胎盘，然后胎膜依次用细镊子解剖。 </li><li>转移解剖胚胎至50ml管中使用的大移液管1×冰冷的PBS。避免直接用钳子拿起胚胎，以尽量减少组织损伤。 </li></ol></li><li> 固定术 <ol><li>固定在10％中性缓冲福尔马林溶液中的解剖胚胎在4℃下超过24小时，用固定剂中的至少10样品体积。 </li></ol></li><li> 预处理 <ol><li>准备结算解决方案:48M尿素，10％甘油，4％十二烷基硫酸钠（SDS），在双蒸水。 </li><li>更换清算解决方案的固定液。 </li><li> 2个星期 - 在室温下为1轻轻转动上的摇杆试样。交易所结算解决方案，一旦在第二和第三天。试样慢慢变得清澈透亮。 </li><li>用10％福尔马林更换结算解决方案和摇杆离开2天。 </li></ol></li><li> 脱水 <ol><li>用1×PBS洗涤预处理样品3次，每次5分钟。 </li><li>脱水在室温下递增乙醇系列上的平缓摇杆样品。为E15.5胚胎中，使用递增的乙醇系列包括50％，60％，70％，80％，85％，90％，95％和100％的乙醇，2小时的每个步骤。对年龄较大的胚胎，培养时间需要增加。 </li></ol></li><li> 染色 <ol><li>准备染色液:补充0.1375克曙红Y二钠盐和0.0275克吖啶橙半（氯化锌）盐至50ml的100％乙醇的。搅拌2小时。用滤纸过滤。在室温下储存，并通过用铝箔覆盖避光。 </li><li>传送标本的染色溶液1小时。 </li><li>用新鲜的染色液和染色一夜之间更换。 </li></ol></li><li> 浸润 <ol><li>准备渗透解决方案:结合百毫升JB-4包埋试剂盒的溶液A，1.25克催化剂的C，0.275克曙红Y钠盐和0.055克吖啶橙半（氯化锌）盐。搅拌混合（200rpm）下，在冰桶2小时，然后用滤纸进行过滤。 </li><li>存放在4℃的渗透解决方案，并通过与铝箔覆盖避光。 </li><li>胚胎转移到渗透液:染色溶液（1:1），并在4℃孵育至少3小时在摇杆。 </li><li>胚胎转移到渗透解决方案，并培育3小时R ON在寒冷的房间摇杆。 </li><li>每天更换一次渗透溶液，并在冷室中留下一个温和摇杆为三天。 </li></ol></li></ol> 2.嵌入<ol><li>保持解决方案B和渗透液冰上。 </li><li>立即嵌入之前，请嵌入（溶液B和渗透液1:25）。 </li><li>东方在嵌入模具样品，然后轻轻地泼冷水解决方案嵌入到嵌入模具。重新定向，如果需要用的销。 </li><li>使用PIN检查嵌入解决方案是否凝固。压出试样块，如果需要修剪。 </li><li>使用单独的模具来获得的横方向重新定向试样块。 </li><li>把块托架上嵌入模具的顶部。轻轻倾嵌入解到模具直到模具和块保持器被嵌入溶液淹没。 </li><li>保持试样在次级容器并​​使其保持在冰上3 - 在通风橱4小时。 </li><li>储存在4℃的固化试样。 </li><li>剥去嵌入模具。放块中的体视显微镜下的斜光来检查在块中的样本的位置。在块面，标志网格线的样品周围。 </li><li>修剪块删除使用标记网格线作为参考包埋材料的访问量。 </li></ol> 3.图像采集<ol><li>夹紧标本块切片机，并获取新的切割面。设置切片厚度（ 如 e9.5-10.5，1.5微米; e11.5-12.5，2.0微米; e13.5-15.5，2.5微米; E16.5-年纪大了，3微米）。保持试样/块在切片机的位置。 </li><li>安装立体变倍显微镜水平面临着样本的块面。 </li><li>打开计算机和显微镜，并启动图像采集软件。 </li><li>可视化和聚焦光学器件的块面在成像软件的"实时取景"模式。 </li><li>如果需要的话，这样的块面是在取景器中央对准显微镜和切片机。 </li><li>调整变焦，以使整个块面是在取景器内。 </li><li>选择绿色荧光蛋白（GFP）的过滤器（激发470±20纳米，分色495纳米，发射525±25纳米），如果需要的话重新聚焦。 </li><li>测量和手动设置曝光时间 （ 例如 80 - 400迷你秒）。 </li><li>获得块面的图像中的每个鲜切后，如果可能，会自动保存图像。 </li><li>包括分辨率，截面厚度和变焦记录的重要参数。 </li><li>完成整个标本，出口和在必要时所有的图像文件转换为JPEG格式之后。 </li><li>倒置使用成像处理软件的所有原始图像，并调整亮度和对比度。 </li><li>保存所有在一个单独的文件夹中处理图像。 </li></ol> 4. 3D可视化<ol><li>上传所有按照指示处理过的图像，三维可视化软件。 </li><li>输入分辨率和截面厚度把所有图像容积数据（ 即，像素的大小），并保存3D文件。 </li><li>对齐使用自动模式下，所有的图像。如有必要，手动调整个别图像。 </li><li>对齐图像保存到一个新的文件名。 </li><li>分析使用三维可视化软件试样的形态特征。 </li></ol>

<ol>
	<li>Gregg, C. L., Butcher, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vivo+imaging+of+embryonic+development:+opportunities+and+challenges.">Quantitative in vivo imaging of embryonic development: opportunities and challenges.</a> Differentiation. 84, 149-162 (2012).</li><li>Liu, X., Tobita, K., Francis, R. J., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+techniques+for+visualizing+and+phenotyping+congenital+heart+defects+in+murine+models.">Imaging techniques for visualizing and phenotyping congenital heart defects in murine models.</a> Birth defects Res C. 99, 93-105 (2013).</li><li>Norris, F. 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=A+coming+of+age:+advanced+imaging+technologies+for+characterising+the+developing+mouse.">A coming of age: advanced imaging technologies for characterising the developing mouse.</a> Trends Genet. 29, 700-711 (2013).</li><li>Weninger, W. J., 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=Phenotyping+structural+abnormalities+in+mouse+embryos+using+high-resolution+episcopic+microscopy.">Phenotyping structural abnormalities in mouse embryos using high-resolution episcopic microscopy.</a> Dis model mech. 7, 1143-1152 (2014).</li><li>Mohun, T. J., Weninger, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Episcopic+three-dimensional+imaging+of+embryos.">Episcopic three-dimensional imaging of embryos.</a> Cold Spring Harb protoc. , 641-646 (2012).</li><li>Sizarov, A., 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=Three-dimensional+and+molecular+analysis+of+the+arterial+pole+of+the+developing+human+heart.">Three-dimensional and molecular analysis of the arterial pole of the developing human heart.</a> J Anat. 220, 336-349 (2012).</li><li>Anderson, R. H., 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=Normal+and+abnormal+development+of+the+intrapericardial+arterial+trunks+in+humans+and+mice.">Normal and abnormal development of the intrapericardial arterial trunks in humans and mice.</a> Cardiovasc Res. 95, 108-115 (2012).</li><li>Huang, Y. C., Chen, F., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarification+of+mammalian+cloacal+morphogenesis+using+high-resolution+episcopic+microscopy.">Clarification of mammalian cloacal morphogenesis using high-resolution episcopic microscopy.</a> Dev Biol. 409, 106-113 (2016).</li><li>Mohun, T. J., Weninger, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embedding+embryos+for+episcopic+fluorescence+image+capturing+(EFIC).">Embedding embryos for episcopic fluorescence image capturing (EFIC).</a> Cold Spring Harb protoc. , 675-677 (2012).</li><li>Weninger, W. J., Mohun, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+analysis+of+molecular+signals+with+episcopic+imaging+techniques.">Three-dimensional analysis of molecular signals with episcopic imaging techniques.</a> Methods mol biol. 411, 35-46 (2007).</li><li>Richardson, D. S., 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=Clarifying+Tissue+Clearing.">Clarifying Tissue Clearing.</a> Cell. 162, 246-257 (2015).</li><li>Hama, H., 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=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat Neurosci. 14, 1481-1488 (2011).</li><li>Susaki, E. A., 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+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157, 726-739 (2014).</li><li>Mohun, T. J., Weninger, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+volume+data+by+episcopic+three-dimensional+imaging+of+embryos.">Generation of volume data by episcopic three-dimensional imaging of embryos.</a> Cold Spring Harb protoc. , 681-682 (2012).</li></ol>

The authors have nothing to disclose.

这里，我们目前用常规的实验室设备收购是快速三维可视化和复杂结构的形态分析兼容串行高分辨图像的修改协议。由于高分辨率图像被从块面，而不是各个部分直接被使用，则细形态特征被保留，并迅速在3D可视化程序数字重建。 3D成像形象化和了解复杂的形态特征提供了显著的优势。大多数3D成像技术依赖于专门的设备1-4。与此相反，EFIC和高分辨成像只利用标准的实验室设备，包括装备有数码相机和切片机14立体显微镜。显微镜和切片机之间的功能耦合使图像采集的自动化功能，整个图像采集过程的自动化。然而，我们已经表明，功能耦合是不是强制性的8。相反，我们只是水平安装体视显微镜，使其直接面对在静止停止位置标准切片机的块面。这个简单的设置的主要限制是高分辨图像必须手动拍摄。我们估计，它需要平均每个图像约5秒。另一个限制是，各个图像可以从一个位置移动到另一个，但这不是一个问题，因为它们是三维重建期间容易重新排列。 使用高分辨技术，我们已经分析了一系列小鼠胚胎的3D图像从E9.5至E13.5的，并发现了由孤胚胎泄殖腔转变成两个独立的结构的新的机制，远侧消化道和下泌尿大片8。后期的高分辨图像质量上演的胚胎是有限的，由于伊红低效渗透（数据未显示）。为了提高组织penetrE15.5和E18.5胚胎的通货膨胀，我们使用两种不同的预处理方法是对荧光成像11兼容并测试新的配方，结算溶液（4M尿素，10％甘油，4％SDS）中。所有这些方法已显示的组织穿透和曙红染色的显著改善。预处理之前和之后的一个重要考虑因素是，试样是容易引起皮肤肿胀或收缩由于渗透压。为了限制渗透压的突然变化，从而减轻组织的变形，我们推荐的解决方案逐渐交换。此外，我们还发现，列入乙醇显著帮助（步骤1.5.4）。期间渗透和嵌入步骤，它从光保持样品并在4℃是重要的。 总的来说，这个协议采用常规的实验室设备获取高分辨图像，这是对于高分辨率三维可视化和形态分析相容。该协议守LD容易地适用于任何标准的实验室设置。

The advent of 3D imaging technologies has opened the possibility of systemic analysis of detailed morphological features during fetal development. A variety of 3D imaging modalities is now available include micro-magnetic resonance imaging (micro-MRI), micro computed tomography, high frequency ultrasound, optical coherence tomography, optical project tomography and episcopic microscopy1-4. Each modality has its own unique features in resolution, contrast, speed, cost, in utero capability and availability.
Episcopic fluorescence image capture (EFIC) and high-resolution episcopic microscopy (HREM) are episcopic microscopy imaging methods4. Here, high-resolution serial images are captured continuously from the block face instead of tissue sections. The resulting images faithfully reflect detailed morphological features with minimal or no tissue distortion. The acquired volumetric data is readily converted to high-resolution 3-D images suitable for accurate morphometric analysis. Because of relative low cost and high resolution, HREM and EPIC have become the superior alternatives for systemic assessment of mouse models of human birth defects2,4-8.
Both EFIC and HREM methods require specimens to be embedded in the suitable medium. EFIC detects autofluorescence emitted from the embedded tissue. Because of this, it is limited to specimens emitting high levels of autofluorescence but not those with relatively weak autofluorescence such as early stage embryos9. To overcome the dependency of autofluorescence, specimens are stained with a fluorescent dye such as eosin for HREM imaging. The choices of dyes and staining methods also make HREM more compatible to detect molecular signals in the context of tissue architecture and morphology4,10.
In this article, we present an improved HREM protocol suitable for whole body assessment of the late stage mouse embryos. To facilitate staining, we include a pretreatment step to increase tissue penetration, thereby enabling HREM imaging of older mouse embryos.

<table><tbody><tr><td>Mice</td><td>The Jackson Laboratory</td><td>C57BL6</td><td></td></tr><tr><td>Ethyl Alcohol</td><td>Pharmco-Aaper</td><td>111000200</td><td>200 Proof, Absolute, ACS/USP/Kosher Grade</td></tr><tr><td>Formalin</td><td>Sigma</td><td>HT501128-4L</td><td></td></tr><tr><td>Urea</td><td>Sigma</td><td>U5128</td><td>Clearing Solution</td></tr><tr><td>Glycerol</td><td>Fisher scientific</td><td>G33-4</td><td>Clearing Solution</td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Invitrogen</td><td>15525-017</td><td>Clearing Solution</td></tr><tr><td>Eosin Y Disodium Salt</td><td>Fisher scientific</td><td>BP241925</td><td>Infiltration Solution</td></tr><tr><td>Acridine Orange hemi (zinc chloride) salt</td><td>Sigma</td><td>A6014</td><td>Infiltration Solution</td></tr><tr><td>Whatman paper</td><td>GE</td><td>3030-153</td><td></td></tr><tr><td>JB-4 Embedding Kit - Solution A</td><td>Polysciences, Inc.</td><td>0226A-800</td><td>Infiltration Solution</td></tr><tr><td>JB-4 Embedding Kit - Solution B</td><td>Polysciences, Inc.</td><td>0226B-30</td><td>Embedding Solution</td></tr><tr><td>JB-4&amp;nbsp;Embedding Kit - Catalyst</td><td>Polysciences, Inc.</td><td>02618-12</td><td>Infiltration Solution</td></tr><tr><td>Embedding Molds</td><td>Polysciences, Inc.</td><td>23185-1</td><td>16 x 8 mm</td></tr><tr><td>Peel-A-Way Sharp Embedding Molds</td><td>Polysciences, Inc.</td><td>18986-1</td><td>22 mm x 22 mm square, truncated to 12 mm x 12 mm</td></tr><tr><td>JB-4&amp;nbsp;Plastic Block Holders</td><td>Polysciences, Inc.</td><td>15899</td><td></td></tr><tr><td>Disposable Graduated Transfer Pipes</td><td>VWR</td><td>414004-014</td><td></td></tr><tr><td>Petrl Dish</td><td>VWR</td><td>25384-088</td><td>Embryo dissection</td></tr><tr><td>Forceps Inox Tip</td><td>&amp;nbsp;ROBOZ</td><td>RS-5015</td><td>Embryo dissection</td></tr><tr><td>Graefe Tissue Forcep</td><td>&amp;nbsp;ROBOZ</td><td>RS-5153</td><td>Embryo dissection</td></tr><tr><td>Delicate Operating Scissor</td><td>&amp;nbsp;ROBOZ</td><td>RS-6700</td><td>Embryo dissection</td></tr><tr><td>14 ml Polypropylene Round-Bottom Tubes</td><td>Falcon</td><td>352059</td><td></td></tr><tr><td>50 ml Polypropylene Conical Tubes</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>Ice Bucket</td><td>Magic Touch Iceware International Corp.</td><td>R19-343</td><td></td></tr><tr><td>100 ml (3.4 oz.) Antistatic Polystyrene Weigh Boats</td><td>VWR</td><td>89106-766</td><td></td></tr><tr><td>330 ml (11.2 oz.) Antistatic Polystyrene Weigh Boats</td><td>VWR</td><td>89106-770</td><td></td></tr><tr><td>Sodium Chloride</td><td>MP Biomedicals</td><td>102892</td><td>PBS Solution</td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P0662</td><td>PBS Solution</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P5405</td><td>PBS Solution</td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Sigma-Aldrich</td><td>S9390</td><td>PBS Solution</td></tr><tr><td>Digital Camera</td><td>Hamamatsu Photonics</td><td>C11440-10C</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2265</td><td></td></tr><tr><td>Illuminator</td><td>Carl Zeiss</td><td>HXP 200C</td><td></td></tr><tr><td>Fluorescence Stereo Zoom Microscope</td><td>Carl Zeiss</td><td>Axio Zoom.V16</td><td></td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Fiber Optic Light Source</td><td>Leica</td><td>KL 1500 LCD</td><td></td></tr><tr><td>Disposable Microtome Blade</td><td>VWR</td><td>95057-832</td><td></td></tr><tr><td>Single Edge Dispenser</td><td>Personna</td><td>62-0330-0000</td><td></td></tr><tr><td>ZEN</td><td>Carl Zeiss</td><td>pro 2012</td><td></td></tr><tr><td>Photoshop</td><td>Adobe</td><td>CS6 v13.0</td><td></td></tr><tr><td>Amira 3D Software</td><td>Visage Imaging</td><td>5.4</td><td></td></tr><tr><td>Computer</td><td>Dell T7600, 2x900GB SAS hard drive and 64 GB DDR3 RDIMM memory</td><td></td><td></td></tr><tr><td>Rocking Platform Shakers</td><td>VWR</td><td>40000-304</td><td></td></tr><tr><td>Standard Hot Plate Stirrer</td><td>VWR</td><td>12365-382</td><td></td></tr><tr><td>Analytical Balance</td><td>Mettler Toledo</td><td>AB54-S</td><td></td></tr></tbody></table>

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.

我们曾报道E9.5和E13.5 8之间的小鼠胚胎的高品质图像HREM。后期阶段胚的图像质量，然而，显著因为荧光染料曙红的有限的组织穿透的损害。为了提高效率，染色，我们测试的是荧光成像兼容11多种预处理方法。具体来说，E15.5小鼠胚胎，用任何的Sca / E A2 12或全身CUBIC 13处理。我们也测试了简化公式，清算溶液（4M尿素，10％甘油，4％十二烷基硫酸钠）。所有的预处理方法提高染色效率。胚胎成为1周结算固溶处理后半透明。在处理后没有检测到明显的组织损伤。预处理的胚胎是embedde之前通过步骤，包括脱水，染色和加工的渗透d。在塑料嵌入媒体。如在第3（图像采集）上述并使用三维可视化软件中显示高分辨图像从嵌入的标本收购。 E15.5胚胎的整个安装面图的一个例子展示了皮肤的详细的形态学特征，包括晶须焊盘和显影毛囊（ 图1）。虚拟剖视图的分辨率是可比的组织切片（ 图2）。 <img alt="图1" src="/files/ftp_upload/54625/54625fig1.jpg" /> 图 1: 在E15.5胚胎的三维表面观这表明皮肤的具体形态特征。发展中的毛囊（小白点）是显而易见的，展示高清晰度的图像。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54625/54625fig1large.jpg" target="_blank">请点击这里查看大图已经</a>这个数字rsion。 <img alt="图2" src="/files/ftp_upload/54625/54625fig2.jpg" /> 图 2: 在E15.5胚胎的中线部分的虚拟剖面视图的内部结构，包括心脏，肝脏，肠道和膀胱都清晰可见。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54625/54625fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy at JoVE.com

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

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) ...

rapid-acquisition-3d-images-using-high-resolution-episcopic

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.6K 次观看.  Wenzhou Medical University. 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.

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

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

科研

生物工程

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

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging organic materials with volumes up to 5 x 5 x 7 mm3 in typical numeric resolutions between 1 x 1 x 1 and 5 x 5 x 5 &#181;m3. Specimens are embedded in methacrylate resin and sectioned on a microtome. After each section an image of the block surface is captured with a digital video camera that sits on the phototube connected to the compound microscope head. The optical axis passes through a green fluorescent protein (GFP) filter cube and is aligned with a position, at which the bock holder arm comes to rest after each section. In this way, a series of inherently aligned digital images, displaying subsequent block surfaces are produced. Loading such an image series in three-dimensional (3D) visualization software facilitates the immediate conversion to digital volume data, which permit virtual sectioning in various orthogonal and oblique planes and the creation of volume and surface rendered computer models. We present three simple, tissue specific protocols for processing various groups of organic specimens, including mouse, chick, quail, frog and zebra fish embryos, human biopsy material, uncoated paper and skin replacement material.

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

We provide simple and robust protocols for processing biopsy material of various species, embryos of biomedical model organisms and samples of other organic tissues in order to permit digital volume data generation with the high-resolution episcopic microscopy method.

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 ...

发育生物学

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

免疫与感染

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

内吞体动力学在生活神经末梢与四维荧光成像可视化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

神经科学

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

研究脊椎动物轴向伸长和分割的三维和四维可视化分析方法

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

细菌细胞的三维成像，实现精确的细胞表现和精确的蛋白质定位

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

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

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

在Cytokineticž圈的活细菌超分辨率成像使用快速三维结构照明显微镜（F3D-SIM卡）

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

三维超F-微丝高分辨率显微镜通过干涉光敏定位显微镜（iPALM）

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

使用微棱镜对头部固定和自由移动的动物进行多层成像

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

作者聚焦:清醒和自由移动状态下神经活动的比较成像

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

在自由移动和头部固定的动物中使用微棱镜对已识别的神经种群进行长期成像

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...