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>

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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?...

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 border="0" cellpadding="0" cellspacing="0" width="1218">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Mice</td>
			<td style="width:356px;">The Jackson Laboratory</td>
			<td style="width:136px;">C57BL6</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Ethyl Alcohol</td>
			<td style="width:356px;">Pharmco-Aaper</td>
			<td style="width:136px;">111000200</td>
			<td>200 Proof, Absolute, ACS/USP/Kosher Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formalin</td>
			<td>Sigma</td>
			<td style="width:136px;">HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td>Sigma</td>
			<td style="width:136px;">U5128</td>
			<td>Clearing Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher scientific</td>
			<td style="width:136px;">G33-4</td>
			<td>Clearing Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate</td>
			<td>Invitrogen</td>
			<td style="width:136px;">15525-017</td>
			<td>Clearing Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin Y Disodium Salt</td>
			<td>Fisher scientific</td>
			<td style="width:136px;">BP241925</td>
			<td>Infiltration Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acridine Orange hemi (zinc chloride) salt</td>
			<td>Sigma</td>
			<td style="width:136px;">A6014</td>
			<td>Infiltration Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Whatman paper</td>
			<td>GE</td>
			<td>3030-153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JB-4 Embedding Kit - Solution A</td>
			<td>Polysciences, Inc.</td>
			<td style="width:136px;">0226A-800</td>
			<td>Infiltration Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JB-4 Embedding Kit - Solution B</td>
			<td>Polysciences, Inc.</td>
			<td style="width:136px;">0226B-30</td>
			<td>Embedding Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JB-4&#160;Embedding Kit - Catalyst</td>
			<td>Polysciences, Inc.</td>
			<td style="width:136px;">02618-12</td>
			<td>Infiltration Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Embedding Molds</td>
			<td>Polysciences, Inc.</td>
			<td style="width:136px;">23185-1</td>
			<td>16x8mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peel-A-Way Sharp Embedding Molds</td>
			<td>Polysciences, Inc.</td>
			<td style="width:136px;">18986-1</td>
			<td>22mm x 22mm square, truncated to 12mm x 12mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JB-4&#160;Plastic Block Holders</td>
			<td>Polysciences, Inc.</td>
			<td style="width:136px;">15899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Graduated Transfer Pipes</td>
			<td style="width:356px;">VWR</td>
			<td style="width:136px;">414004-014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petrl Dish</td>
			<td style="width:356px;">VWR</td>
			<td style="width:136px;">25384-088</td>
			<td>Embryo dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps Inox Tip</td>
			<td style="width:356px;">&#160;ROBOZ</td>
			<td style="width:136px;">RS-5015</td>
			<td>Embryo dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Graefe Tissue Forcep</td>
			<td style="width:356px;">&#160;ROBOZ</td>
			<td style="width:136px;">RS-5153</td>
			<td>Embryo dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Delicate Operating Scissor</td>
			<td style="width:356px;">&#160;ROBOZ</td>
			<td style="width:136px;">RS-6700</td>
			<td>Embryo dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">14 ml Polypropylene Round-Bottom Tubes</td>
			<td style="width:356px;">Falcon</td>
			<td style="width:136px;">352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml Polypropylene Conical Tubes</td>
			<td style="width:356px;">Falcon</td>
			<td style="width:136px;">352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ice Bucket</td>
			<td style="width:356px;">Magic Touch Iceware International Corp.</td>
			<td style="width:136px;">R19-343</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mL (3.4 oz.) Antistatic Polystyrene Weigh Boats</td>
			<td style="width:356px;">VWR</td>
			<td style="width:136px;">89106-766</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">330 mL (11.2 oz.) Antistatic Polystyrene Weigh Boats</td>
			<td style="width:356px;">VWR</td>
			<td style="width:136px;">89106-770</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Sodium Chloride</td>
			<td style="width:356px;">MP Biomedicals</td>
			<td style="width:136px;">102892</td>
			<td>PBS Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Potassium Chloride</td>
			<td>Sigma</td>
			<td style="width:136px;">P0662</td>
			<td>PBS Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td style="width:136px;">P5405</td>
			<td>PBS Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Sodium phosphate dibasic heptahydrate</td>
			<td style="width:356px;">Sigma-Aldrich</td>
			<td style="width:136px;">S9390</td>
			<td>PBS Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Digital Camera</td>
			<td style="width:356px;">Hamamatsu Photonics</td>
			<td style="width:136px;">C11440-10C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Microtome</td>
			<td style="width:356px;">Leica</td>
			<td style="width:136px;">RM2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Illuminator</td>
			<td>Carl Zeiss</td>
			<td>HXP 200C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Fluorescence Stereo Zoom Microscope</td>
			<td>Carl Zeiss</td>
			<td style="width:136px;">Axio Zoom.V16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Stereo Microscope</td>
			<td>Olympus</td>
			<td style="width:136px;">SZX16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Fiber Optic Light Source</td>
			<td style="width:356px;">Leica</td>
			<td style="width:136px;">KL 1500 LCD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Disposable Microtome Blade</td>
			<td style="width:356px;">VWR</td>
			<td style="width:136px;">95057-832</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Single Edge Dispenser</td>
			<td style="width:356px;">Personna</td>
			<td style="width:136px;">62-0330-0000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">ZEN</td>
			<td>Carl Zeiss</td>
			<td style="width:136px;">pro 2012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Photoshop</td>
			<td style="width:356px;">Adobe</td>
			<td style="width:136px;">CS6 v13.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Amira 3D Software</td>
			<td style="width:356px;">Visage Imaging</td>
			<td style="width:136px;">5.4</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:377px;">Computer</td>
			<td style="width:356px;">Dell T7600, 2x900GB SAS hard drive and 64 GB DDR3 RDIMM memory</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Rocking Platform Shakers</td>
			<td style="width:356px;">VWR</td>
			<td style="width:136px;">40000-304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Standard Hot Plate Stirrer</td>
			<td style="width:356px;">VWR</td>
			<td style="width:136px;">12365-382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Analytical Balance</td>
			<td style="width:356px;">Mettler Toledo</td>
			<td style="width:136px;">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?...

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

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

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

造血干细胞的自动定量 - 在脱钙骨的组织学图像间质细胞的相互作用

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

科研

发育生物学

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 ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

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 ...

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 ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, and 3D reconstruction.

使用全载免疫作用化学和三维重建对咽弓动脉的可视化与分析

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

4D显微镜:使用Nomarski显微镜揭示 秀丽隐杆线虫 胚胎发育

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...