这项研究导致这些结果根据出让协议已收到的资金来自欧盟第七框架计划（FP7/2007-2013）号码n。 228334和241526。这一研究项目也已支持人类前沿科学计划的研究经费（RGP0027/2009），以及由意大利教育部教育，大学和研究中的旗舰项目NANOMAX的框架，并通过在意大利的框架卫生部"干细胞提案征集"。这项研究已经进行了的图标地基"恩特卡萨迪Risparmio Di Firenze酒店"支持的研究活动的框架。

1。组织准备和储存<ol><li>通过标准穿心灌注13固定鼠脑用20毫升0.01M的磷酸盐缓冲盐水（PBS）溶液中，接着在0.01M的PBS 100毫升多聚甲醛4％。仔细调节两种溶液的pH至7.6:pH值稍低的值可淬EGFP的荧光。 </li><li>在多聚甲醛4％，4℃后固定切除大脑过夜</li><li>冲洗两次（每次30分钟）在0.01 M的PBS，并存储在0.01 M的PBS于4℃ </li></ol> 2。脱水及结算所注意:该协议用 ​​于脱水和结算紧跟由Becker 等 10所描述的。 <ol><li>从脱水溶剂（四氢呋喃，THF）中，用的是色度的强烈淬灭XFP荧光，过滤的THF与碱性活性氧化铝（活性级布罗克曼我，THF中的约250克/升）除去过氧化物tography列。避免柱的开裂，这会降低过氧化去除。 </li><li>加稳定剂（二丁基羟基甲苯，250毫克/升）的最终溶液中，即使在THF中在过滤之前已经稳定。稳定剂，其实是由三氧化二铝保留:不加稳定剂THF可发展为大量的过氧化物，可易爆危险。 </li><li>脱水整个鼠脑在纯水中的渐变系列THF中的50％，70％，80％，90％，96％，100％，每1小时，然后100％过夜。将小瓶用样品上的旋转轮。为了避免光线照射，包装用铝箔的小瓶。 </li><li>过滤清洗溶剂（二苄基醚，DBE）与氧化铝（基本活性，活性级布罗克曼我，DBE约250克/升）用过滤漏斗。 </li><li>清除100％DBE的样品。后3和6小时改变溶液。 </li></ol> 3。试件安装<ol><li>当大脑通过眼睛（典型看起来透明第二DBE变化后新增Cally 1小时），它安装在尖成像板通过下列方法之一: </li><li><ol><li> 直接安装 -轻轻地投身于其中窍门（ 图3a）的样品。 </li><li> 琼脂糖盘 -跳水上的提示做的6％琼脂糖凝胶光盘。轻轻地固定在琼脂糖光盘用丙烯酸胶水的样品（ 图3b）。等待约1分钟进行固化。 </li><li> 琼脂糖烧杯 -扎拉上的尖 ​​端制成的3％琼脂糖凝胶的烧杯中。轻轻地放置在样本上的烧杯的底部（ 图3c）。 </li></ol></li></ol>注1:安装在琼脂糖烧杯中应首选时，它是重要的影像的全部样品，但是，周围的标本琼脂糖凝胶产生了额外的光散射这可能会损害图像质量。如果样品的一小部分可以被排除在成像时，最好装入规格imen琼脂糖磁盘或直接在小费。前者的方法是最适合养脑水平（从成像脑的腹侧或背侧部除外），后者以保持大脑垂直（从脊髓的成像嗅球的一部分或不包括）。 注2:在琼脂糖凝胶应在水中来制备，然后脱水，用THF（50％和100％4小时各）和DBE 100％清除4小时或直到它看起来清晰透明。 <ol start="3"><li>安装试样以下的上述方法之一后，使用镊子将样品室内部定位成像板。与结算解决方案填补了腔。 </li></ol> 4。体层摄影术准备<ol><li>除去缝隙中收集尽可能多的荧光越好。 </li><li>照明样品低激光功率，以防止漂白。 </li><li>移动样品中使用的软件驱动的微定位系统·三个方向米。确定每一方向上的最小和最大坐标的量，样品被照射并在视摄像机（ 图4a）的视场内。 </li><li>将上述坐标作为"预断层扫描"子程序的参数。还指定相邻堆叠之间的断层摄影中使用和在z方向上的前体层摄影术采样步骤的距离。 </li><li>开始预层析，收集的图像中的每个堆叠以在（4.4）中指定的Z采样。 </li></ol>注意:所收集的图像被用于通过软件来制备试样形状和位置（ 图4b），其允许限制断层扫描采集仅由样品实际占用的体积，减少成像时间，从而漂白粗略地图。 5。断层扫描采集<ol><li>将样品用微定位系统的光片的外侧。 </li><li>公司REASE激光功率，并停止所有的扫描系统的运动，以便照亮只有固定线在视场的中心。 </li><li>安装缝隙，并使用从结算解决方案的自体荧光信号对齐。你应该清楚地看到在视场中心的切割线。 </li><li>重新启动扫描系统，降低激光功率，并使用微定位系统的样品移动光片的内部。注意，与狭缝中使用的激光功率会比没有它用于更高，因为空间滤波器块不可忽略的光的分数。 </li><li>调整的幅度和控制软件的扫描和反扫描系统的偏移，直到图像看起来清晰明亮。 </li><li>运行断层扫描采集软件，设置实验中适当的z步骤之后。该卷将被拍摄了许多平行的，部分重叠的叠层（ 图5a），图像将被保存在分层文件夹结构。 </li></ol> 6。拼接和可视化<ol><li>使用自动化软件（ 图4c）与合成黑色的图像（所收集的相同类型和尺寸的图像，即那些-，但具有零强度）完成所获取的体积。此步骤是必须的，为了使拼接软件来处理一个完整的立方体体积。 </li><li>启动Vaa3D软件（从免费的下载<a href="http://www.vaa3d.org/" target="_blank">http://www.vaa3d.org/</a> ）安装TeraStitcher和TeraManager的插件。 </li><li>加载TeraStitcher插件。选择包含​​成像体积的目录中，指示坐标轴的相对方向（相对于一个参考右手坐标系统）和体素尺寸。 </li><li>启动拼接的第一部分。该软件将计算堆栈夫妻之间的相对位移，并找到一个全局最优placem耳鼻喉科的所有堆叠在一起（ 图5b）。 </li><li>选择保存缝合量在单分辨率或在多分辨率格式。后者能够为多分辨率可视化与TeraManager插件。在这两种情况下，如果分辨率更高的图像比几GB的，还可以选择多堆栈保存方式，并指定个别substacks的大小。这将使高效地访问所存储的数据。 </li><li>启动拼接的第二部分。该软件将合并对齐堆栈，并将它们保存在单或多栈模式（ 图5c和5d）。在年底关闭TeraStitcher插件。 </li><li>加载TeraManager插件。与多堆叠多分辨率音量选择文件夹，并注明体素的尺寸和轴的方向。该卷将在最小分辨率被加载。 </li><li>要放大到一个更高的分辨率，选择使用鼠标右键单击modalit的一个里程碑Vaa3D的Y，然后放大使用鼠标滚动。或者，您也可以直接选择投资回报率来放大与右键单击，或指定感兴趣的体积的坐标。 </li><li>要放大到较低的分辨率，只需用鼠标滚动。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

激光共聚焦显微镜加上化学清算代表一个功能强大的工具来研究标记的荧光探针，无论是蛋白质或合成染料整个老鼠大脑的神经解剖学。由于焦平面以外发出的光被一个物理空间滤波器，高对比度的成像是可能的也是厚厚的标本，同时保持高帧率典型的轻质板材建筑。 主要的问题来处理使用所描述的方法时，与是的对比度最大化。实际上，可用的信号都通过化学和光学因素降低。从化学角度来看，基于有机溶剂的清关程序可以从荧光蛋白和其他荧光染料7解渴排放。作为溶剂，过滤以除去过氧化物，如由Becker 等人 10，允许保持荧光的良好水平也是在溶剂被清除的组织。发布编辑水性结算方法14不降低荧光效率，反而会导致透明度较差，与整个鼠大脑处理，至少在非线性光学技术。 在另一方面，在目前有有长 ​​工作距离的高折射率（n≈1.56）结算使用的解决方案纠正任何商业目的。因此，在该样品浸入介质和物镜的设计1之间的折射率失配产生了强烈的球面像差。除了减少显微镜的分辨率，这样的像差导致的下降的图像亮度和对比度时，由于光被铺展在更广阔的领域。可能的解决方法对这个问题包括使用自适应光学系统15和/或静态非球面校正器。 任何改善图像的对比度和亮度也很容易影响到成像的速度，因为在短每幅图像tegration时间可以选择。此刻CLSM可以承受大约10 6微米3 /秒的成像速度，以100毫秒9的照相机的曝光时间。在此速度下需要从1-3天到图像的整个鼠脑，这取决于样品的大小。虽然在图像质量没有显著下降在整个这么长的成像会议的光片照射6固有的低光漂白主要是因为，观察到的，很长一段时间需要像一个鼠标的大脑可以在实践中减少CLSM的适用性。一个10倍的系统效率，加上采用高速摄像头进行检测，将减少成像时间为几个小时，让日常使用所描述的协议。 尽管所有的上述局限性，激光共聚焦显微镜成像耦合到组织许可证化学结算重建整个老鼠大脑与微米级分辨率，在不到一个一周。其他光学方法全脑成像能力在较长的准备和/或成像时间2-4的价格，或稀疏Ž采样5的分辨率比激光共聚焦显微镜高，但。 本文中介绍的方法可以在与荧光标记不同的策略组合使用:转基因动物表达在选定的神经元亚群，病毒转染，脑实质内注射染料等荧光蛋白在实践中，动物牺牲前所有染色战略与激光共聚焦显微镜兼容。事实上，整个老鼠大脑中的验尸报告荧光标记，未能证实到现在，无论如何，如果此类染色将有可能在不久的将来，可与激光共聚焦显微镜重建标本的数量将变得更大，可能包括人脑组织。 总之，在使用与TIS组合共聚焦光片显微镜起诉清算和多分辨率的数据管理可以使研究人员重建和分析宏观标本在微米级分辨率，与那些在好大多数实验室的手技改力度。激光共聚焦显微镜的潜在应用当然不限于神经系统科学，但涵盖所有中，轻质板材镜已被使用的研究领域，如胚胎发育16,17和小动物18的解剖。

相较于我们的细胞和分子机制基本脑功能的了解，我们的优良神经解剖学的知识，看起来还处于起步阶段1。事实上，我们还缺乏长期预测中的神经元胞体或整个大脑的位置综合地图。事实上，许多技术努力致力于重建小鼠大脑与微观的分辨率。根据树脂包埋标本，连续切片的光学方法，刀口扫描显微镜（KESM）2，微光学切片层析成像（MOST）3和荧光MOST（fMOST）4，可提供亚微米的三维解析成像整个鼠标的大脑。然而，所需要的单个样品的制备和成像的总时间是几个星期的数量级，限制了这种方法的实际应用。基于连续切片的另一项技术（但没有树脂包埋），串行双光子断层扫描（STP）5，允许高三维高分辨率成像。然而，该技术已被证明在整个小鼠脑中只用非常稀疏采样，采集1段每50微米5，并以我们的知识STP没有产生又一个鼠大脑的完整采样重建。 在高速重建整个标本的三维尖端的光学方法是轻质板材镜6。在该光学方案的样品被照射的薄光片和荧光发射是沿垂直于所述照明平面的轴线收集。以这种方式仅在焦荧光被激发，这是能够实现宽视场配置的光学切片，以保证高的帧速率。当联接到清除基于水的折射率匹配液7的取代的协议，光片显微镜已经被应用到宏观标本，作为整个老鼠大脑8。然而，样品诱导的光散射，这会影响甚至清除标本，降低了光片导致的离焦的荧光团的激发这增加了整体的模糊所获取的图像。 有选择地收集仅在对焦光子，我们最近耦合光片照射到激光共聚焦检测方案9。在共焦光片显微镜（CLSM），出焦和散射光子被一个线性空间滤波器（狭缝）在检测之前（ 图1）拒绝。为了实现在光片建筑共焦操作时，该照明用的扫描线的装置产生，而在检测路径的去扫描系统创建在狭缝的位置的激发扫描线的静态图像。第三扫描系统重建的二维图像上的相机传感器。激光共聚焦显微镜能提供在清理鼠标100％对比度增强同时保持10赫兹帧频的大脑相对于传统光片镜。用激光共聚焦显微镜能够重建整个老鼠大脑为2μm的XY和9微米的Z轴分辨率，并具有足够的对比度，以识别单个荧光标记的神经元。 在本文中，我们描述了实验管道重建小鼠的大脑与激光共聚焦显微镜。醛固定小鼠的大脑梯度脱水系列的无过氧化物的四氢呋喃，并随后被清除在无过氧化物的二苄醚（ 图2），下面通过Becker 等人 10的清零标本开发的协议，然后小心地安装在一尖板放置在一个特制的激光共聚焦光片显微镜成像室。样品可以直接安装通过在板（ 图3a）的前端切入它;备选地它可以清除琼脂糖凝胶上的盘（ 图上被粘图3b），或放置在作出清零琼脂糖凝胶（ 图3c）的烧杯中的空腔中。然后进行整个大脑的光学断层扫描，因为被收购的许多平行相邻堆覆盖所有的脑容量（ 图4）。预断层扫描采样，产生的样本位置和形状的粗略地图，保证只在由样本所占的体积进行成像。该断层栈随后缝合在一起使用自制软件，并保存在一个多分辨率分层方案11。老鼠大脑最终可以探索与原型多分辨率谷歌地图般的可视化软件。这两个软件工具集成为开源平台Vaa3D 12插件。 最终推导出的小鼠大脑的代表图像来演示在研究小鼠精的neuroanatom描述实验管道的能力年。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Phosphate Buffered Saline Tablets</td>
 <td>Sigma-Aldrich</td>
 <td>P4417_100TAB</td>
 <td></td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Agar Scientific</td>
 <td>R1018</td>
 <td></td>
 </tr>
 <tr>
 <td>Peroxide-free Tetrahydrofuran</td>
 <td>Sigma-Aldrich</td>
 <td>186562</td>
 <td></td>
 </tr>
 <tr>
 <td>Aluminum oxide activated, basic, Brockmann I</td>
 <td>Sigma-Aldrich</td>
 <td>199443</td>
 <td></td>
 </tr>
 <tr>
 <td>Dibenzyl ether</td>
 <td>Sigma-Aldrich</td>
 <td>108014</td>
 <td></td>
 </tr>
 <tr>
 <td>Butylated hydroxytoluene</td>
 <td>Sigma-Aldrich</td>
 <td>W218405 </td>
 <td></td>
 </tr>
 <tr>
 <td>Agarose Type III-A, High EEO</td>
 <td>Sigma-Aldrich</td>
 <td>A9793</td>
 <td></td>
 </tr>
 <tr>
 <td>Acrylic glue</td>
 <td>Bostik</td>
 <td>D2498</td>
 <td></td>
 </tr>
 <tr>
 <td>Vaa3D software</td>
 <td>open source</td>
 <td></td>
 <td>freely downloadable from <a href="http://www.vaa3d.org/" target="_blank">http://www.vaa3d.org/</a> </td>
 </tr>
 </tbody>
</table>

micron-scale resolution optical tomography of entire mouse brains with confocal light sheet microscopy

理解哺乳动物大脑的结构在单细胞分辨率是神经科学的关键问题之一。然而，在整个大脑的神经元映射胞体和预测仍是成像和数据管理技术的挑战。事实上，宏观的卷需要具有高分辨率和对比度重建在合理的时间，生产的TB级范围内的数据集。我们最近展示了一种光学方法（光共聚焦显微镜片，激光共聚焦显微镜），能够获得标记与增强型绿色荧光蛋白（EGFP）整个老鼠大脑的微米级重建。结合光板照明和激光共聚焦检测，激光共聚焦显微镜可以让深层成像内宏观清除标本具有高对比度和速度。在这里，我们描述了完整的实验管道获得标有荧光蛋白整个老鼠大脑的全面和人类可读的影像。结算及安装procedures进行了描述，连同步骤通过获取许多平行相邻的堆叠在其整个体积中进行光学断层摄影术。我们发现开源定制软件工具使多个栈和多分辨率的数据导航的拼接使用。最后，我们示出脑映射的一些例子:从L7-GFP转基因小鼠，其中所有的浦肯野细胞的选择性标记，以及从THY1-GFP-M鼠标，其特征在于由一个随机稀疏神经元标记的全脑小脑。

所描述的协议可以被用来重建与微米级的分辨率无论是整个老鼠大脑或部分切除，而不需要物理切片。作为代表性的结果，在图6中示出了L7-GFP的小鼠（10产后天）的整个小脑。在这种动物的所有浦肯野神经元都贴有绿色荧光蛋白。如果我们放大，小脑皮层的典型的层状结构可以看出（ 图7）。进一步放大可清楚区分每浦肯野细胞胞体（ 图8）。所描述的协议，因此可用于筛查神经元空间组织各种神经发育研究。 作为第二个代表性的结果，我们从成年THY1-GFP-M鼠标unsectioned大脑呈现的图像。在此转基因动物的EGFP表达在一个随机稀疏神经元的子集。大脑的右半部分示出在图9中。正如我们放大式越走越（ 图10和图11），神经元突起成为区分。这一结果表明，所描述的协议允许微米级分辨率成像在整个成年小鼠的大脑，开启在神经退行性疾病的小鼠模型细胞高分辨率学习全脑解剖结构的可能性。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/50696/50696fig1.jpg" /> 图1。该设备的共焦光片显微镜（CLSM）的光学方案。（一）顶视图，显示了激励途径。从488nm的二极管泵浦固态（DPSS）激光器的激光发射，扩展和准直由第一望远镜后，进入一个声光可调谐滤波器（AOTF），其调节光束的强度。然后，第二望远镜进一步扩大了光束。一个电流计镜垂直（沿y）的扫描是<html>该装置时，集中由试样腔室内部的透镜。（二）的侧视图，表示该检测途径。光由检测物镜收集被垂直通过一个电流计镜退扫描并通过透镜聚焦，从而产生移动励磁线路的固定图像。在固定的图像的位置的线性空间滤波器（狭缝）放置。狭缝后，检流计反射镜放置在4023透镜系统内重建一个2维图像的电子倍增电荷耦合器件（EM-CCD）的芯片上。一种荧光带通滤波器取出检测之前的所有激励的杂散光。激励，检测和带通滤波检测光代表射线是由蓝色，淡蓝色和绿色的光线，分别描绘。在显微镜下实际使用的所有镜头的焦距表示。 <a href="https://www.jove.com/files/ftp_upload/50696/50696fig1large.jpg" target="_blank">点击这里查看大图</a> 。 jove_content"FO:保持together.within页="总是"&gt; <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/50696/50696fig2.jpg" /> 图2试样的结算。甲醛固定小鼠的大脑，其在4℃下保存于PBS中的0.1M（a）中 ，脱水的梯度系列的四氢呋喃（THF，b）和随后在100％的二苄基醚清零（DBE ，C）。脱水步骤也导致一些组织收缩。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/50696/50696fig3.jpg" /> 图3。样品安装的激光共聚焦显微镜成像的清脑可以直接暴跌在放倒安装板（一），粘在清除琼脂糖盘（b）中 ，或在清除琼脂糖烧杯（三）插入。 图4。断层扫描采集。预断层常规样品含有脑（a）确定所需的图像中的所有样品平行相邻的堆的数量和长度的平行六面体（b）所示 。断层扫描后，生成黑色图像来完成虚拟购入量为平行六面体（c）所示 。 <img alt="图5" fo:content-width="4in" src="/files/ftp_upload/50696/50696fig5.jpg" /> 图5。卷拼接的图像采集栈对方（一）之间的部分重叠，使他们与TeraStitcher插件（二）精确对准。该插件然后合并对齐栈在任何一个单一的堆叠（三）或多站cked（D）的多分辨率表示。 <img alt="图6" fo:conteint-width="5in" src="/files/ftp_upload/50696/50696fig6.jpg" /> 图6。一个P10 L7-GFP的小鼠，其中所有的浦肯野神经元都贴有绿色荧光蛋白。比例尺，0.1毫米的全小脑 。 <img alt="图7" fo:content-width="4in" src="/files/ftp_upload/50696/50696fig7.jpg" /> 图7。放大在小脑的一部分，如图6所示，示出了小脑皮层，比例尺，500微米的典型层状结构 。 <img alt="图8" fo:content-width="4in" src="/files/ftp_upload/50696/50696fig8.jpg" /> 图8。进一步放大在图6中所示的小脑的一部分的。浦肯野细胞胞体可以明确区分编辑。比例尺，200微米。 <img alt="图9" fo:content-width="5in" src="/files/ftp_upload/50696/50696fig9.jpg" /> 图9。大脑从成年人THY1-GFP-M鼠标，其特征在于由一个稀疏非特异性神经元的EGFP标记。比例尺，1毫米右半部 。 <img alt="图10" fo:content-width="4in" src="/files/ftp_upload/50696/50696fig10.jpg" /> 图10。放大，在图9所示的半脑的一部分的，示出了海马和。比例尺，200μm以下的皮层的一部分 。 <img alt="图11" fo:content-width="4in" src="/files/ftp_upload/50696/50696fig11.jpg" /> 图11。进一步放大，在图9所示的半脑的一部分的几个轴突在皮层能够清楚地区分。比例尺，50微米。

Watch this Scientific Journal Video about Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy at JoVE.com

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

在这篇文章中，我们描述了完整的实验程序来重建，具有分辨率高，荧光标记的小鼠大脑的优良大脑解剖。所描述的协议包括样品制备和清算，样品安装成像，数据后处理和多尺度可视化。

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

micron-scale-resolution-optical-tomography-entire-mouse-brains-with

Research

JoVE Journal

Neuroscience

16.6K 次观看.  European Laboratory for Non-linear Spectroscopy (LENS). 在这篇文章中，我们描述了完整的实验程序来重建，具有分辨率高，荧光标记的小鼠大脑的优良大脑解剖。所描述的协议包括样品制备和清算，样品安装成像，数据后处理和多尺度可视化。 

视频: Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

科研

神经科学

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

用光片荧光显微镜观察运动轴突导航和小鼠胚轴突树枝状的定量化

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

小鼠大脑的自由漂浮免疫染色

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the ...

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a cascade of events leading to neurotransmitter release. Moreover, changes in presynaptic calcium levels mediate the activity of many intracellular proteins and play an important role in synaptic plasticity. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The neuromuscular junction is a suitable model for studying synaptic plasticity, as it has only one type of neurotransmitter. This article describes the method for loading a calcium-sensitive dye through the cut nerve bundle into the mice's motor nerve endings. This method allows the estimation of all parameters related to intracellular calcium changes, such as basal calcium level and calcium transient. Since the influx of calcium from the cell exterior into the nerve terminals and its binding/unbinding to the calcium-sensitive dye occur within the range of a few milliseconds, a speedy imaging system is required to record these events. Indeed, high-speed cameras are commonly used for the registration of fast calcium changes, but they have low image resolution parameters. The protocol presented here for recording calcium transient allows extremely good spatial-temporal resolution provided by confocal microscopy.

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

使用共聚焦显微镜以高时间分辨率记录小鼠神经肌肉接头中的钙瞬变

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a ...

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature vibrations of molecules. However, these vibrational signatures are also characteristic of molecules within anatomical tissue such as the brain, making it increasingly useful and applicable for Neuroscience applications. For example, CARS can measure lipids by specifically exciting chemical bonds within these molecules, allowing for quantification of different aspects of tissue, such as myelin involved in neurotransmission. In addition, compared to other techniques typically used to quantify myelin, CARS can also be set up to be compatible with immunofluorescent techniques, allowing for co-labeling with other markers such as sodium channels or other components of synaptic transmission. Myelination changes are an inherently important mechanism in demyelinating diseases such as multiple sclerosis or other neurological conditions such as Fragile X Syndrome or autism spectrum disorders is an emerging area of research. In conclusion, CARS can be utilized in innovative ways to answer pressing questions in Neuroscience and provide evidence for underlying mechanisms related to many different neurological conditions.

Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices

Visualizing myelination is an important goal for many researchers studying the nervous system. CARS is a technique that is compatible with immunofluorescence that can natively image lipids within tissue such as the brain illuminating specialized structures such as myelin.

相干反斯托克斯拉曼光谱（CARS）在脑切片中成像髓鞘形成的应用

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

使用MRI，组织清除和光片显微镜对完整的新生小鼠大脑进行全脑单细胞成像和分析

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or presbycusis is a primary cause of sensorineural hearing loss and is characterized by damage to hair cells, spiral ganglion neurons (SGNs), and the stria vascularis. These structures reside within the cochlea, which has a complex, spiral-shaped anatomy of membranous tissues suspended in fluid and surrounded by bone. These properties make it technically difficult to investigate and quantify histopathological changes. To address this need, we developed a light-sheet microscope (TSLIM) that can image and digitize the whole cochlea to facilitate the study of structure-function relationships in the inner ear. Well-aligned serial sections of the whole cochlea result in a stack of images for three-dimensional (3D) volume rendering and segmentation of individual structures for 3D visualization and quantitative analysis (i.e., length, width, surface, volume, and number). Cochleae require minimal processing steps (fixation, decalcification, dehydration, staining, and optical clearing), all of which are compatible with subsequent high-resolution imaging by scanning and transmission electron microscopy. Since all the tissues are present in the stacks, each structure can be assessed individually or relative to other structures. In addition, since imaging uses fluorescent probes, immunohistochemistry and ligand binding can be used to identify specific structures and their 3D volume or distribution within the cochlea. Here we used TSLIM to examine cochleae from aged mice to quantify the loss of hair cells and spiral ganglion neurons. In addition, advanced analyses (e.g., cluster analysis) were used to visualize local reductions of spiral ganglion neurons in Rosenthal&#39;s canal along its 3D volume. These approaches demonstrate TSLIM microscopy&#39;s ability to quantify structure-function relationships within and between cochleae.

A light-sheet microscope was developed to image and digitize whole cochlea.

使用光片荧光显微镜对老化耳蜗进行成像

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or ...

小鼠视网膜 OCT 纤维图与共聚焦图像的比对

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

作者聚焦：用于改善青光眼诊断和治疗的体内和离体视网膜成像的进展 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

可见光光学相干断层扫描光纤图与同一小鼠视网膜的共聚焦图像的对齐

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

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Subtitles

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像