我们感谢C.霍耶的手稿的批判性阅读，B.宾格尔（基因技术公司）参与剧本的叙述，边北路-LY与尾静脉注射外源凝集素染料的共享提高了血管成像的经验，和M Solloway（Genentech公司），用于在胰腺成像中使用的小鼠。 GFP-M线被创造了由J.萨内斯（华盛顿大学圣路易斯分校）和CX3CR1-GFP小鼠由R.利特曼（N​​YU）。这项工作的支持，Genentech公司

所有动物实验均按照对小鼠〜3-5月龄IACUC（机构动物护理和使用委员会）的规定执行。作者宣称没有竞争的财务权益。 1，动物灌注和组织准备时间:30-60元鼠标+后期固定分（数小时至过夜）。 <ol><li>称取动物和麻醉用氯胺酮（80-200毫克/公斤）和甲苯噻嗪（7-20毫克/千克）或2.5％阿佛丁（0.5 ml/25 g体重的IP）。 </li><li>等待几分钟麻醉取完的效果。 </li><li>捏动物的趾部和尾部，以确保动物是完全麻醉。 </li><li>用0.1M磷酸缓冲液（PB）或0.1M的磷酸盐缓冲溶液（PBS）中5-10分钟，第一灌注动物在RT直到血液完全从组织中移除。 </li><li>切换灌注固定液:4％PFA在0.1M PB（或0.1 M的PBS），并继续灌注用4％PFA中以3毫升/分钟的速度30-40分钟。 </li><li>解剖感兴趣的器官/ s的不小心损坏， 例如 ，避免穿刺和挤压正在被解剖的组织。 </li><li>除去周围的器官（结缔组织，脑膜或硬脑膜物）的培养皿中填充有PBS的额外的组织。 </li><li>后固定在4％PFA器官的几个小时或过夜，4ºC。避免长时间后固定，因为PFA可能淬灭信号或增加autofluorecense加班10尤其是对GFP通道（这是在红色和远红色通道较小的问题）。 </li><li>刚开始清算程序前用PBS在室温洗器官2-3倍。 </li></ol> 2，组织清算时间:2-3小时的小器官和1-4天为大器官。 注:该组织的荧光标记通过转基因表达，病毒转染，染料追踪或反机身标签应清除之前完成。在室温下执行所有组织清算的步骤。结算解决方案，焦磷酸钾，DBE，BABB（1份苯甲醇和2部分苯甲酸苄酯）和二氯甲烷的毒性，应避免吸入（在通风良好的通风橱内进行实验），并与皮肤直接接触（使用丁腈手套）。 <ol><li>制备50％（体积/体积），70％，和在单独的玻璃瓶如下蒸馏水80％十二烷基硫酸钠的稀释:对于50％的十二烷基硫酸钠， 例如 ，混合25ml THF溶液和25ml的蒸馏水在一个玻璃瓶体积比50毫升大。 </li><li>轻轻摇动瓶子1-2分钟混合的解决方案。 </li><li>清楚地标记玻璃瓶。 </li><li>盖紧的瓶子，并保持工作的解决方案在一个黑暗的内阁。为获得最佳效果，请不要使用相同的解决方案，工作时间超过1-2周。因此，责令清算解决方案，提供给能够使用新的股票试剂的最小体积。 </li><li>填补玻璃小瓶（ 如 2-3毫升），与第一结算解决方案，50％的焦磷酸钾，并从PBS器官转移到玻璃瓶中，开始清算。 </li><li>盖紧玻璃小瓶用自己盖，并使用一个旋转器（ 例如 ，一个轮子搅拌器）进行搅拌。 </li><li>用铝箔覆盖，并保持玻璃小瓶在黑暗中。开始旋转体搅拌的玻璃小瓶中所指示的时间（ 表1）的样品以恒定的速度（〜30转）。 </li><li>除去交换溶液和添加在该协议下一个时，在表1中的时间就完成了。 </li><li>收集清理废为被保存在通风橱的玻璃废物容器。 重复在协议中每个结算解决方案前面的步骤，直到结束。使用新的巴斯德吸管当一个新的结算溶液中加入（ 例如 ，从THF转变为DBE）。 </li><li>在最后的清除步骤与BABB或DBE，孵育时间可以延长或SHortened直到样品变得在可见光完全透明的（或淡黄/透明，如果它是一个非常大的组织像大脑）。 </li><li>保持清除器官在最后结算解决方案，在任何时候，包括在成像步骤。 </li></ol> 3，准备清除器官成像时间:5-15分钟。 注:图片的清除器官一旦一个完整的信息交换的实现。为此，请按照下面的步骤来准备样品光薄片显微镜（ 例如 ，超高倍显微仪）或共聚焦/多光子显微成像。 注:清除器官将失去随着时间的推移其荧光强度（尤其是荧光蛋白如 GFP，抗体标记更耐磨，甚至可以提供更好的结果具有较长的孵育）。对于成像，各种荧光显微技术可以，只要被用作器官的适当处理的CLEA环溶液来实现。 <ol><li>对于光片显微镜<ol><li>放置或安装适当的样品。 </li><li>通过手动转动样品支架4的螺钉固定的清除器官。 </li><li>样品浸入thelight表显微镜填充有BABB或DBE，取其被用来在最终结算步骤的成像室（优选玻璃制的）。 </li></ol></li><li>对于多光子/激光共聚焦显微镜<ol><li>安装在成像滑动（或成像室）与最终的成像溶液中的样品，以能够与图像的多光子或共焦显微术，其通常使用的油或水浸入目标。 </li><li>用牙科水泥，使周围组织的边界。 </li><li>与BABB或DBE填补了池牙科水泥得到完全刚性之前。 注:牙科水泥快速固化;练习几次，以熟悉它试图在实际样品之前。 </li><li>立即将在池的中间，用玻璃盖覆盖它的清样。 </li><li>按玻璃盖，直到它完全由牙科用粘固剂的密封和接触的清除器官，这将允许通过共聚焦/多光子显微术在达到最大成像深度的表面上。 注:这将确保没有结算解决方案将在成像过程中溅出。避免直接浸渍成像透镜进入结算解决方案，这可能会损害镜头，除非它是耐BABB / DBE或有保护盖。 </li></ol></li></ol> 4，成像清除器官时间:15-45分钟。 <ol><li>在该显微镜可以提供最佳的分辨率收集Z扫描覆盖整个组织清除（如果使用的镜头允许的话）。 </li><li>放大感兴趣的区域，收集更高分辨率的图像。 </li></ol>该数据5。考试与软件阿米拉<ol><li>加载图像意甲s到软件阿米拉与ResolveRT模块。 </li><li>在"图像读取参数"窗口，然后按确定输入正确的像素大小。 </li><li>选择"多刨视图"子应用程序。然后用"厚度"滑块来选择最佳的阈值的灰度值，调整2D和3D的值。 </li><li>通过浏览切片在不同的二维取向和利用农作物角模块在3D视图中可视化的样品。 注:该协议的详细故障排除指南可以在埃蒂尔克等 4被发现。 </li></ol>

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A., Murphy, C. S., Hazelwood, K. L., Davidson, M. W., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+and+photostable+photoconvertible+fluorescent+protein.">A bright and photostable photoconvertible fluorescent protein.</a> Nat Methods. 6, 131-133 (2009).</li></ol>

没有什么可以透露。

组织信息交换的方法的目标是通过在清除器官匹配不同的组织层的折射率，以减少成像光的散射。其结果，成像光可以穿透深入到组织中，刺激标记的细胞/分子。组织清理17这个老概念已经深入人心越来越多的关注技术对完整的老鼠大脑中的第一个成熟的应用程序通过DODT和同事2后。从那以后，有一个快速成长的新的结算方法，包括3DISCO，爱生雅/ E，ClearT2，清晰度和SeeDB 3,4,7,18-20列表。从本质上讲，它们都旨在通过保留荧光标记来呈现透明的器官深层组织成像。其中，3DISCO脱颖而出作为最直接的和可重复的方法之一。这是比较快的比较（分别为1-5天，而2-3周为成人的大脑，）上述其他结算方法21。它已经被成功完全适用于不同的器官，如脑，脊髓，淋巴结，脾，肺，实体瘤，胰腺和乳腺3,4,9。此外，一些出版物由独立的研究小组已经用这种方法来获得成像效果22,23。然而，不同的组织清算程序可能是有利的条件不同。例如，当的Sca / e和SeeDB适用最好在胚胎组织中6,7，它们是水基的结算方法，这可能是更容易在成像过程中进行处理。相比之下，清晰度是一个复杂和昂贵的结算方法。但它可以是有利的抗体标记的情况下，特别是在大型组织如脑8。 以从3DISCO最好的成像效果最关键的一步就是组织标签，这是该组织清算之前完成。至关重要的是从最强的荧光信号成为可能。这可以bE在各种方式，包括荧光蛋白的转基因表达实现的，通过合成染料，染病毒或抗体标记跟踪。但应记住的是，任何结算过程将减少组织中的荧光信号。因此，如果荧光信号不够强的开头- 即它是不容易检测到的结算成像的组织清除前将提供糟糕的结果。 有清正在等待改进方法的一些限制。一个重要的限制是，由于清除试剂改变清除器官的结构，它们不能在活组织中。因此，实验如与光活化mEos2 24应固定并清算之前进行。清算后，由光明和耐光蛋白质的荧光信号的超分辨成像成为可能。另外，因为信息交换协议削弱了荧光蛋白的强度，则清除器官不能存储长时间。要注意，一些器官更难被清除是很重要的。特别是，如果解剖的器官保存大量的血细胞（ 如脾，肝），他们是比较难以清除和形象。它也是相对更难实现一个完整的清理大型组织，如成年啮齿类动物的大脑。这样的组织，可能需要较长的培养时间，这可能，另一方面，降低了荧光信号。此外，显微镜方法的发展，可以像大型透明机关立即在高分辨率是等待需要解决的问题。该技术的另一个重要的限制是组织标记。虽然通过遗传或病毒表达强烈的荧光信号是理想的，不是每一个细胞或分子可以通过基因或病毒标记。在另一方面，厚组织抗体标记不是一个简单的过程，甚至不是宝ssible在大多数情况下。它可能需要特殊的通透协议和潜伏期较长时间（数天至数周）3。同样重要的是要记住，该组织主要是清除由于脱水和脱脂后收缩〜20％，在每一个维度（测定脊髓组织）。均成比例发生这种收缩并应在量化步骤进行校正。所观察到的在所呈现的结果中，荧光标记的结构保持不变，这是蛋白质的完整性在清除组织的指示。因为最终清除组织的疏水性质的，它不能被进一步抗体染色或嵌入在含水溶液如 Focusclear -这是在净度，或80％的甘油。最后，它是分析成像数据的压倒性的大小是一个挑战。例如，当整个鼠脑进行扫描，可在细胞的分辨率，这将是非常难以追踪和量化所需的STRuctures（ 例如 ，神经元或神经胶质细胞网络），并比较在不同的样品中的自动化方法。总之，尽管研究人员的目标，找出新的结算方法，上述（清不同组织的难度，成像在高分辨率的较大的区域，以及复杂的数据分析）的各种挑战，应并联改善。 总之，最近开发的组织清算技术，包括3DISCO，优雅地表明，完整器官的高分辨率三维成像成为可能。使用这些方法，科学家可以在完整的器官获得的细胞和分子的解剖学信息。在透明的器官，这些开拓性的三维成像研究激发了越来越多的研究人员破译复杂的解剖和组织/器官的健康和疾病的分子组织。

生物组织的组织学检查是了解分子/细胞的性质在它们的自然背景下的根本途径。理想情况下，整个器官的高分辨率成像是最翔实和期望。因为光具有有限的穿透深度，因散射1，固定机构具有为了进行高分辨率显微成像进行切片。因此，大多数的组织学研究在几个组织切片代表器官，只有一小部分被执行。在一些研究中， 例如，那些旨在描绘出在大脑或脊髓神经细胞的连接，从靶器官的所有组织切片被收集并成像的三维重建。然而，组织切片及个别段落的后续成像有各种限制。这些措施包括为费时，并导致组织的一个不完整的三维重建，由于机械变形和困难IES中所产生的图像的对准。 近日，结算及成像完整透明的器官已经发展成为一个显著的解决这个缺点2,3。后结算，整个器官呈现透明，允许成像光的旅行端-端（ 图1）使用激光扫描显微镜诸如多光子或光片的显微镜，以产生unsectioned器官的高分辨率的图像（ 图2）。各研究组已经开发了新的组织信息交换的协议，以便能够像他们感兴趣的用于不同目的的组织。这些措施包括有机溶剂2-5，基于8清算协议水中6,7和电泳。其中，溶剂的3维成像清除器官或3DISCO是在多种生物样品，包括中枢神经系统（CNS）的器官，免疫器官和实体瘤的一种容易应用的协议。在ADDIT离子，它可以与诸如光片荧光显微镜（LSFM），多光子及共聚焦激光扫描显微镜不同的显微技术相结合。 3DISCO是基于与现成的和便宜的试剂，如四氢呋喃（THF）和二苄基醚（DBE）4清零。整个协议可以采取短至3-4小时。因此，3DISCO是一个强大的和快速的技术相比，可能需要几个星期到几个月才能完成9传统的组织学方法。

<table border="0" cellpadding="0" cellspacing="0" width="913">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:201px;">Thy-1 GFP (GFP-M) mouse</td>
			<td style="width:248px;"></td>
			<td style="width:175px;"></td>
			<td style="width:291px;">can be obtained from Jackson&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CX3CR1 GFP mouse</td>
			<td></td>
			<td></td>
			<td>can be obtained from Littman lab at NYU</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TgN(hGFAP-ECFP) mouse</td>
			<td></td>
			<td></td>
			<td>can be obtained from Jackson&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde&#160;</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Na2HPO4&#160;</td>
			<td>Mallinckrodt</td>
			<td>7917-16</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaH2PO4&#160;</td>
			<td>(Mallinckrodt, 7892-04)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-2-2 Tribromoethanol</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Methyl-2-butanol</td>
			<td>Sigma</td>
			<td align="right">152463</td>
			<td>used to prepare Avertin solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline&#160;</td>
			<td>Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrahydrofuran&#160;</td>
			<td>Sigma</td>
			<td>186562-12X100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichloromethane&#160;</td>
			<td>Sigma</td>
			<td>270997-12X100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dibenzyl ether&#160;</td>
			<td>Sigma</td>
			<td>Sigma, 108014-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol&#160;</td>
			<td>Sigma</td>
			<td>305197-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate&#160;</td>
			<td>Sigma</td>
			<td>B6630-250ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lectin FITC&#160;</td>
			<td>Sigma</td>
			<td>L0401-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CC10</td>
			<td>Santa Cruz</td>
			<td>SC-9772</td>
			<td align="right">0.388888889</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin&#160;</td>
			<td>APP pharmaceuticals&#160;</td>
			<td align="right">504001</td>
			<td>used in perfusion&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass vials&#160;</td>
			<td>VWR</td>
			<td>548-0554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps&#160;</td>
			<td>FST</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini Rotator&#160;</td>
			<td>VWR</td>
			<td>100498-878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum Foil&#160;</td>
			<td>Fisherbrand</td>
			<td>01-213-104</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100mL Media Storage Bottles</td>
			<td>Kimax Media Bottles</td>
			<td>EW-34523-00</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Minipuls evolution perfusion pump&#160; with MF4* medium flow pump head&#160;</td>
			<td>Gilson, Inc</td>
			<td>F110701 and F117606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscopy slide</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastWell</td>
			<td>Grace Bio-Labs</td>
			<td>FW20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slip</td>
			<td>Corning</td>
			<td>2940-245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass petri dish&#160;</td>
			<td>Corning Pyrex</td>
			<td>3160-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dental cement&#160;</td>
			<td>Lang Dental</td>
			<td>1223PNK</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Quantofix Peroxide Test Strips</td>
			<td>Sigma</td>
			<td align="right">37206</td>
			<td></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:201px;">Amira with RT resolve microscopy module 
			&#160;</td>
			<td>Visage Imaging</td>
			<td>image analysis software</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:201px;">Imaris</td>
			<td>Bitplane imaging</td>
			<td>image analysis software</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ&#160;</td>
			<td>NIH</td>
			<td>freeware</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging cleared intact biological systems at a cellular level by 3disco

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

神经元是高度极化的细胞具有极长的形态。其功能取决于它们之间的相互之间以及与其它组织中形成的连接。因此，映射神经元的结构组织是非常重要的，以便了解他们在健康和疾病如何运作。然而，追踪整个神经系统，最近被任命为connectomics 11如此巨大的神经元连接-仍然在神经科学中最困难的挑战之一。为此，无论是欧盟12和美国13已经启动大项目来映射人类大脑。 虽然3DISCO可对多种脏器被采用，它一直特别有用追查脊髓和大脑长的神经连接。例如，使用大清零脊髓节段的超高倍显微仪扫描时，轴突的连接可以遵循以上在啮齿类动物脊髓（厘米图2）。以类似的方式，在整个清除小鼠大脑（ 图3a）和海马（ 图3b）可以被成像到跟随在大脑中神经元的连接。 当共聚焦或多光子显微镜上使用清除器官，成像分辨率可显著改善，特别是在z维度。例如，从GFP-M线的小鼠（ 图4a）清零脊髓多光子成像实现整个脊髓中的整个深度（〜1.5-2毫米）的无缝图像。在清除脊髓共聚焦显微术提供了以类似的方式（ 图4b和c）改进的分辨率。清除大脑的多光子显微成像提供了非常高清晰度的图像可视化的神经元结构的细节，包括树突棘（ 图5）。该样品3DISCO可以标以不同的方式，包括转基因表达，VIR人染，染料跟踪和抗体标记。例如，有可能使用标记的凝集素结合的荧光示踪剂4脑（ 图6a和b）和脊髓（ 图6c和d）中，它可以被用来研究血-脑屏障（BBB的整个脉管系统在健康和疾病）。 两个小胶质细胞和星形胶质细胞是高度牵连的神经退行性疾病的病理包括老年痴呆症的疾病和外伤14,15。使用3DISCO，它们的密度和分布在脊髓（ 图7）或脑进行研究。 非神经组织也可以成像。例如克拉拉细胞在整个啮齿动物肺部可免疫标记的抗体和成像未剖开在单细胞水平（ 图8）。同样地，也能够以清除和图像的细胞，例如α细胞中的unsectioned胰腺组织（ 图9）。 <img alt="图1" fo:content-width="4in" src="/files/ftp_upload/51382/51382fig1highres.jpg" width="500" /> 图1。3DISCO组织清算呈现透明的深部组织成像unsectioned组织。所看到的可见光未清除（a）和清除（ 二）脊髓组织。结算后，深部组织的激光扫描显微镜成为可能。 （ 三）未清除和清除脊髓组织是由双光子显微成像。比例尺在A，B = 0.5毫米，C = 100微米。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51382/51382fig2highres.jpg" width="500" /> 图2。脊髓跟随轴突扩展3DISCO成像。从THY-1 GFP转基因小鼠系（GFP-M）的解剖脊髓被分成更小的部分（〜4mm）的。以下结算协议小组织（ 表1）后的透明脊髓采用超高倍显微可视化。在水平了〜4毫米脊髓段的三维重建（ 一 ），冠状（b）和矢状面图（c）。 （四）代表跟踪的轴突（红色）显示在灰度透明视图。所指示的区域中（D）（E）高倍镜视野。比例尺在A，B，C，D = 0.5毫米，E = 20微米。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51382/51382fig3highres.jpg" width="500" /> 清除脑及海马的图3。3DISCO成像。清除大脑和GFP-M小鼠海马的例子进行成像与超显微镜。整个大脑（a）和海马（二 ）证明神经元netwo三维可视化RKS在被成像的透明组织。在一个= 2毫米和b中比例尺= 20微米。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51382/51382fig4highres.jpg" width="500" /> 图4。组织清算增强了多光子共聚焦显微镜获得的分辨率。由多光子（a）或共聚焦显微镜成像GFP-M透明小鼠脊髓节段（B，C）。注意，清除基本上提高了分辨率和成像深度揭示轴突和树突的精细结构。比例尺在= 100微米，在B，C = 20微米。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51382/51382fig5highres.jpg" width="500" /> 图5。3DISCO成像树突棘。由多光子成像GFP-M透明小鼠脑组织。扫描的皮层区域的版本呈现3D视觉效果tical（a）和水平方向的角度（b）所示 。 （三）〜从所指示的水平的50微米的突起（A，B）。第（三）证明神经元的结构，包括树突棘（箭 ​​头）的细节（四）高倍率指示的区域。比例尺在= 50微米，在B，C = 25微米，在D = 5微米。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51382/51382fig6highres.jpg" width="500" /> 图6。血管3DISCO。要标记的血管在整个器官，外源凝集素-FITC染料灌注（结算前）中所描述的16使用。值得注意的是，一提的是，我们发现，尾静脉注射示踪剂并可对示踪剂的心脏灌注更好的信号。收集固定大脑和脊髓后，他们被清除，我通过超高倍显微仪马吉德。整个小鼠脑血管中的热图的三维可视化（a）和灰度级（a）所示 。 （二）在三维热图的小鼠脊髓段（c）和灰度级（D）的脉管的可视化。基于外源凝集素染色的强度中产生的热图;蓝色:低强度（背景）和红色:高强度（血管）。比例尺在一个= 2毫米，B = 1毫米，并且在C，D = 250微米。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51382/51382fig7highres.jpg" width="500" /> 神经胶质细胞清除中枢神经系统组织的图7。3DISCO。在小胶质细胞TGH（CX3CR1-EGFP）或星形胶质细胞TGN（hGFAP-ECFP）表达绿色荧光蛋白转基因小鼠的脊髓被清除，并用多光子显微镜成像。小胶质细胞的3D渲染被示为表面体积的可视化（a）和透视图（ <sTRONG&gt; B）。 （三），一种光学投影（〜50微米）呈现，以显示在脊髓小胶质细胞的细节。星形胶质细胞的3D渲染被示为表面体积的可视化（a）和透视图（b）所示 。 （F）一种光学投影（〜50微米）呈现，以显示在脊髓的星形胶质细胞的细节。比例尺在A，B，D，E = 100微米，在C，F = 50微米。 <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/51382/51382fig8highres.jpg" width="500" /> 图8。3DISCO一整个安装肺叶与Clara细胞的抗体染色。对于肺叶全贴抗体染色，10周龄的BALB / c雌性小鼠，经右心室用PBS以灌注从肺部清除血液。肺充气后用4％PFA和固定的O / N在室温固定液日量至少三次Ë组织。第二天，肺简要地在PBS中漂洗和右叶被放于5ml PBS中1％的Triton X-100进行透化48小时或直至组织沉没。所有染色均在0.2％的Triton X-100进行的在PBS中含有5％FBS和2％BSA和漂洗均在0.2％的Triton X-100的PBS中。用抗-CC10染色是72小时，在4℃，接着广泛洗涤约6小时。初级抗体进行荧光标记，用抗山羊的Alexa Fluor-594第二抗体过夜，4℃，接着广泛洗涤6小时到过夜来实现。翌日，染色瓣通过50％，70％，80％十二烷基硫酸钠被清除为30分钟各由三个洗涤30分钟，100％十二烷基硫酸钠，随后20分钟的DCM，随后在DBE 30分钟。比例尺在a和b = 1毫米，C = 100微米。 <img alt="图9" fo:content-width="5in" src="/files/ftp_upload/51382/51382fig9highres.jpg" width="500" /> 数字<html> 9，胰腺3DISCO，如在协议上述小鼠灌注后，胰腺解剖和清除的短协议。荧光是由ROSA26 LSL tdTomato重组诱导他莫昔芬治疗背着一个200 kb的BAC转基因跨度内源性胰高血糖素的鼠标轨迹与CreERT2插入胰高血糖素的ATG小鼠得出。在箭头标记一些荧光标记α细胞在胰岛的。比例尺中，b和c = 1毫米，D = 500微米。 <img alt="表1" fo:content-width="6in" src="/files/ftp_upload/51382/51382table1.jpg" width="600" /> 表1。组织清算协议的各种组织。组织清算协议，为不同组织的例子。注意，清除时间为每个步骤可以被缩短，或者根据需要，以提高信息交换性能延长。小组织的大致权重是〜20-100毫克和大脑〜300-500毫克。TTPS :/ / www.jove.com/files/ftp_upload/51382/51382table1.jpg"目标="_blank"&gt;请点击这里查看此表的一个更大的版本。

Watch this Scientific Journal Video about Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO at JoVE.com

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

成像在细胞水平上通过3DISCO清除完整生物系统

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact ...

imaging-cleared-intact-biological-systems-at-cellular-level

Research

JoVE Journal

Neuroscience

26.4K 次观看.  Genentech, Inc.. To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen. 

视频: Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

依赖国家对TMS的:一看动机幻视行为

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

科研

神经科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

高分辨率人类中脑功能磁共振成像方法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

多光子显微镜的清除小鼠脑组织表达YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

微透析的乙醇操作性乙醇的自我管理和乙醇的测定气相色谱

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

脑植入的微型器件及周围组织的完整的组织学特性研究

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

利用微流体装置进行机械刺激和高分辨率成像的研究C.线虫

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

使用荧光宏镜检查,通过"完整小鼠头骨"进行脑脊液传输的 Vivo 成像

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...