The authors have nothing to disclose.

这项研究进行了按照批准的医学科学，伊朗大学（德黑兰，伊朗）伦理委员会提出的指南护理和使用实验动物（NIH）的政策。 1.动物，手术和标本准备注:准备脑组织的三维共聚焦显微镜分析。 <ol><li>分割动物随机分为两组:Aβ1-40 -注射（N = 8），和Aβ1-40 -注射用染料木黄酮处理（N = 8）;手术前施用染料木素（1​​0mg / kg的稀释在车辆中，如聚乙氧基化蓖麻油）灌胃1小时。 </li><li>麻醉动物用氯胺酮（100毫克/千克）和赛拉嗪（10毫克/千克）。捏脚趾后，确认正确的麻醉缺乏撤退反射的。在手术过程中使用软膏的眼睛，以防止干燥。 </li><li>将动物立体定位仪和剃的头。应用IODINE溶液清洗切口前的头皮和手术过程中保持操作现场无菌，以减少感染的风险。海马注射Aβ1-40立体定向（4微升）在-3.5毫米后到前囟，±2毫米横向中线和-2.8毫米以下硬脑膜，根据大鼠脑图谱8。对于立体定位方法请参见上一公布9。 </li><li>提供术后观察/护理，直至动物充分意识到，以确保动物的舒适性。保持动物温暖的恢复过程中，不把它们返回到与其他动物的笼子，直到完全康复。手术后，在动物的注意任何感染的迹象。 </li><li>氯胺酮（150毫克/千克）手术三周后深麻醉动物。通过灌注用0.9％生理盐水动物其次是4％多聚甲醛的0.1M PBS（pH值= 7.4）执行穿心固定，然后取出并在10所描述修复大脑&lt;/ SUP&gt;。 </li><li>在4℃后固定在4％多聚甲醛的大脑2-3天。 </li><li>嵌入石蜡块组织通过使用组织灌封设备，以及避免下填充或过度填充盒，因为它可能与正确对准或切片干扰。注意在石蜡块中的组织的方向。 注:大鼠海马，例如，靠近大脑半球的后部。因此，组织应该嵌入的方式，切片开始于脑的后部。使用Paxinos地图集8为指导，以达到所期望的解剖结构。 </li><li>将切片机远离空气气流或门道，因为空气流动做出艰难的部分的处理。 </li><li>使用部分的厚度≥20微米，因为这与深度是必要的，以产生完整的星形胶质细胞的Z-堆栈的图像。不要使用第一对夫妇的部分，因为他们可能有不想要的厚度由于热ēxpansion。 </li><li>放置部分温水（5±2 C，低于熔化温度为石蜡）刚好足够长的平坦化部分的表面上。 注:在膨胀部分的会干扰结构的形态。用微小的画笔仔细地转移部分。收集二点五十八每张幻灯片上的部分。 </li><li>在直立位置的齿条商店滑动并让它们在37℃干燥几个小时或O / N。 </li></ol> 2，免疫组化<ol><li> Deparaffinize切片在二甲苯中，2×10分钟，和再水合通过醇梯度（99.8％，95％，70％，每10分钟）和PBS。 </li><li>孵育抗胶质纤维酸性蛋白（GFAP）稀释1:1500于PBS含0.25％牛血清白蛋白（BSA），0.25％的Triton X-100和3.5％正常血清（O / N，在4℃）。 </li><li>在PBS中进行3×5分钟洗涤部分。 </li><li>孵育次级碱性磷酸酶共轭antibodieS表示1小时（1:100，21 C，在PBS中稀释）。 </li><li>在PBS洗的章节3×5分钟。 注:为二级抗体后，用正确方法洗手的部分，以减少背景染色是很重要的。 </li><li>孵育在黑暗的部分与液体永久红显色（15-20分钟）。注意:使用前准备的解决方案不超过30分钟。 </li><li>在PBS中进行3×5分钟洗涤部分。 </li><li>最后，染色4-6二脒基-2-苯基吲哚的核（DAPI; 1:500，在PBS中稀释）之前以覆盖打滑。镜前存放在冰箱24-48小时的幻灯片。 </li></ol> 3.共聚焦显微镜注:对于定量评价（见下文），选择一个星形胶质细胞具有清晰可见DAPI染色细胞核以最小的重叠枝，以减少错误的风险。生产的Z-Stack图像非常耗时。要有耐心，并且在加工过程中不停止。立式激光共聚焦显微镜为used将星形胶质细胞创建3D图像。 <ol><li>放置在显微镜阶段滑动，并选择63X物镜。 </li><li>打开成像软件后点击Start系统。点击定位标签。去分配，并选择GFP来调整合适的光。 </li><li>打开快门，以反射光。找到反射左轮手枪右侧的快门，并选择应该由星形胶质细胞（绿色，红色或紫色）被反射的颜色;红色（FSet20 WF）在这里被使用。 注意:不要忘记在显微镜的目镜直接聚焦的画面。 </li><li>为了找到最佳的焦点，点击全封闭，放在屏幕模式显微镜针，然后单击采集卡上。 </li><li>点击采集卡在智能安装程序;打开一个窗口。从列表中选择合适的荧光团（ 即 DAPI和Alexa Fluor 555，在当前项目中）。颜色是自动选择。 </li><li>点击最佳的信号，并应用，然后点击设置曝光;计算机将自动设置曝光参数。 </li><li>为了优化图像，进入通道窗口。取消标记TRACK2，突出Track1和点击直播。如果需要的话聚焦影像。 </li><li>在通道窗口调整下面的键;点击1AU自动优化针孔。这是很重要的是要做到这一步，否则共焦图象将是最优的。调整增益有最好的强度（保持低于800此设置，以减少额外的噪声）。最后，设置数字增益2和3之间。 </li><li>取消标记TRACK1，点击TRACK2并重复步骤3.8 TRACK2。然后停止直播。 </li><li>进入采集模式窗口。提高图像通过优化帧大小和平均。为了改变框的大小去X * Y，并选择1024×1024。选择一个缓慢的速度来创造一个更好的形象。 </li><li>去平均，然后选择一个数≥4。这个平均值表示将被平均，以全国生产的扫描数Ë获得的图像。现在点击捕捉按钮，保存拍摄的2D图像。 </li><li>对于3D影像，在智能设置标记的Z-Stack项目造成的Z-Stack窗口中打开。 </li><li>设置的Z堆叠的第一和最后位置，即细胞的深度。首先点击直播。然后用显微镜的聚焦驱动集中星形胶质细胞在组织中的最上位置，其中的Z-Stack是开始。点击组第一。然后向下集中在组织，其中Z-Stack的扫描，应停止星形胶质细胞的最低位置。单击设置最后。然后停止直播。 </li><li>选择时间间隔; 1.01微米用在目前的研究;选择的时间间隔可以通过点击最优设置的片数来进行。 </li><li>点击开始实验。保存图像，一旦扫描完成。这个图像将被用于下列参数的测量:表面积和星形胶质细胞领土体积，整个胶质细胞包括胸罩nches，细胞体，和细胞核。 </li><li>获得使用10X或20X物镜按照步骤3.6-3.11所述2D图像。这个图像可以被用于测量抗体的荧光免疫反应性，和星形胶质细胞密度（个/ 1000微米2）的强度。 </li></ol> 4，星形胶质细胞密度和胶质纤维酸性蛋白+荧光强度<ol><li>打开20X 2D图像。在图像窗口左侧点击组织相容（Hisogram）。 </li><li>为了计算星形胶质细胞的密度，选择在20X目标三连冠的视野。算显示出一个清晰的体躯具有最小，重叠在田里分支的星形胶质细胞的数量。 </li><li>量化荧光强度中，选择使图像窗口的页脚一个合适的工具（矩形或圆形），并选择的区域进行测定。注意该图像下的平均值和标准偏差自动出现。在目前的研究中，在0.7毫米2的STR之间的矩形区域iatum和叶片内侧齿状回用。 </li></ol> 5.星形胶质细胞分行<ol><li>量化分支的长度和总数目，使用中的图像解析软件3D图像（具有20X获得）。沿着选定的星形胶质细胞的每个分支的长度手工画一条线。然后选择测量工具专门用于测量线的长度。为了量化分支的数量，选择计数工具计数标注的项目数。 </li></ol> 6.体积和表面积注:在3D图像分析软件手动标记的结构，需要重点和培训。在开始实验前练习多次。 <ol><li>量化星形细胞细胞核，躯体，单元主体和领土的体积和表面积，3D图像（在步骤3.1-3.16收购）转移到一个3D图像分析软件如Volocity 6.1。 </li><li>打开软件，并创建一个新的图书馆。将所有图像向左灰色列中的新库。然后，选择一个3D图像。在右边，有不同的信道用不同的颜色。打开一个感兴趣信道;例如测量细胞核时，蓝色通道的DAPI。手动更改亮度，以获得最佳的对比度。 </li><li>选择工具菜单，去进行音量，并产生一个单一的3D图像了Z堆栈图像。点击属性，在编辑菜单，可以肯定的是X，Y和Z属性显示;否则是不可能进行三维分析。 </li><li>点击工具栏上的手绘RIO工具图标。为了测量星形胶质细胞核的体积和表面积周围画DAPI标记的核的线。 </li><li>为了测量星形胶质细胞体的体积和表面积周围绘制胞体不含分支的线。 </li><li>为了测量整个星形胶质细胞（细胞体含分支机构）的体积和表面积周围画苏摩下面的一行各分公司的表面。 </li><li>为了评估星形胶质细胞领土的体积和表面积画树枝的顶端之间的一条线。 注:按键盘上的Delete删除不必要的图纸。 </li><li>点击在图像窗口创建测量协议的顶部测量菜单上;打开包含不同的工具的窗口。 </li><li>拖动查找对象到窗口（窗格）的顶部。举一个描述性的名称，人口1，选择相关的颜色，然后点击测量。打开另一个窗口。 </li><li>选择一个参数，即 ，体积或表面积。单击确定。该数据将显示在表格中。 </li><li>保存测量菜单上的数据单击并选择使测量项目。 </li><li>从打开的窗口中被称为一个新的测量项目。然后给一个名称的数据，然后点击确定。 </li><li>最后，出口数据统计软件如图形垫或Excel程序。使用t检验来分析数据，然后绘制2D克APHS。 </li></ol>

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这项工作是由郡议会东约特兰省的，（瑞典）和细胞分子研究中心授予在医学大学伊朗（德黑兰，伊朗）的支持。

在目前的协议，我们采用三维激光共聚焦形态来评估与星形胶质细胞形态进行了相关的12个不​​同的参数。为了这个目的，大鼠Aβ1-40的海马组织-诱导的星形胶质细胞增生，有或没有染料木素预处理作为抗炎剂被使用。通过使用3D图像和形态的软件，我们能够表明染料木黄酮对星形胶质细胞增生，即星形胶质细胞形态的影响。 变化的脑组织的细胞内和细胞外环境中可以改变体积细胞/组织的和/或大小。这些改变是在不同的脑损伤3,11考虑病理特征。为了量化这些变化中，使用了多种技术。大多数调查是基于使用，让只有行政长官的一小部分信息通过光或电子显微镜（EM）拍摄的2D低分辨率图像LL。为了克服这种限制，对高清晰度的图像三维共焦形态被开发11。共焦显微镜的另一个优点是，一个单元的体系结构可以自同时的Z-Stack成像进行几个连续的二维图像得到被研究。此外，3D共聚焦显微镜提高通过过滤背景噪声11，以改善图像的质量的可能性。 这里介绍的方法是费时;采取的Z-Stack图像共聚焦显微镜需要几分钟的时间对每个单体电池。此外，手动标记在软件的结构需要一些实践，研究者可能需要重新进行绘图几次，以确保该结构被正确地标记。应当指出，在三维图像分析软件如Volocity的"魔"标签，可用于自动标记在单个图像的所有DAPI染色细胞核。可以使用此选项时，星形胶质细胞进行培养。大脑切片，然而，不仅含有星形胶质细胞，但也有许多其他的细胞，如小胶质细胞和神经元。这意味着许多的DAPI染色细胞核的不属于星形细胞。与星形胶质细胞相关的核应使用具有两个DAPI标记的细胞核和GFAP免疫反应阳性星形胶质细胞的图像进行手动标记。 在这个协议中，抗GFAP用于显现在石蜡切片星形胶质细胞。星形胶质细胞，也可以通过使用其它抗原12,13，或转基因小鼠可视化。研究者应了解的方法的限制，并使用基于其目的可能的最佳实验设计。使用抗GFAP的限制，例如，是可以被检测到只有10-15％的细胞骨架。这种限制，但是，可以研究星形胶质细胞增生，当成为一种优势;星形胶质细胞的数量增加，并在作为密集网络的星形胶质细胞的分行trogliosis可以使它难以识别单个细胞如果检测到小区的一个更大的区域。尽管这一协议描述了12种不同的参数进行量化，但研究者可以选择适合自己目标的人。根据我们的经验，所有参数的测量可以提供有关被显著影响在一定的条件下，或由一个处理参数的有价值的信息。在共焦显微镜拍摄的3D图像执行的量化方法也可在冰冻切片上进行。这个问题是治疗样品（固定，脱水，包埋和切片）以类似的方式，因为这些步骤可导致收缩组织的或肿胀。 最后，三维共焦成像，在与形态软件组合，使得可以量化与细胞的形态相关联的若干参数。我们在这里介绍的协议可以用于形态学瓒的评价中出现的细胞，在不同的病理条件下，或作为干预策略的效果GES。

在健康的中枢神经系统（CNS），星形胶质细胞发挥 ​​血流量，能量代谢，突触功能和可塑性，和细胞外离子的调节中起重要作用的神经递质和动态平衡1-3。此外，星形胶质细胞，以不同的有害刺激和异常时，例如通过反应性星形胶质细胞增生其特点是肥大，增殖和星形胶质细胞4,5-功能重塑外伤，感染，局部缺血或神经变性响应。 反应性星形胶质可以设计在组织中的炎症反应和修复过程，因此，可能会影响治疗性干预的临床结果。因此，星形细胞已经在过去几十年，作为治疗性干预的各种影响CNS疾病的潜在目标接收到的关注神经学家。 星形胶质细胞通常有一个星状的形状，以及-Defined分支中传遍了SOMA 6。在大脑中的疾病状况，星形分支成为旋绕和显示肿胀的端部7中，例如在淀粉样蛋白β（Aβ）的存在。 本文介绍了协议分析由共聚焦显微镜获得的星形胶质细胞的三维图像。测量了每个星形细胞十二个不同的量化参数:所述表面区域和星形胶质细胞的领土（覆盖有星形细胞的组织），整个细胞（包括分支），细胞体，和细胞核的体积;的总长度和分支的数目;用于星形细胞抗体检测的荧光强度;和星形胶质细胞的密度（个/ 1000微米2）。为了这个目的，我们使用脑切片从大鼠暴露于海马内注射Aβ1-40的含或不含染料木黄酮处理作为抗炎物质。所描述的协议可以用于形态的alysis 在体外或体内在不同条件下的不同细胞类型。

<table><tbody><tr><td>Amyloid beta 1-40</td><td>Sigma Aldrich</td><td>79793</td><td>Keep in -70 &amp;deg;C</td></tr><tr><td>Genistein</td><td>Sigma Aldrich</td><td>446-72-0</td><td>keep in -20 &amp;deg;C</td></tr><tr><td>polycolonal rabbit antibodies against glial fibrillary acidic protein</td><td>DAKO</td><td>Z0334</td><td></td></tr><tr><td>alkaline phosphate-conjugated swine anti-rabbit IgG antibodies</td><td>DAKO</td><td></td><td></td></tr><tr><td>Liquid Permanent Chromogen</td><td>DAKO</td><td>K0640</td><td></td></tr><tr><td>Liquid permanent Red Substrate Buffer</td><td>DAKO</td><td>K0640</td><td></td></tr><tr><td>Cremophor EL</td><td>Sigma Aldrich</td><td>27963</td><td>Polyethoxylated castor oil - Step 1.1</td></tr><tr><td>LSM 700 Confocal Laser Scanning Microscopy</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Volocity 6.3</td><td>Perkin Elmer Inc.,</td><td></td><td></td></tr><tr><td>Image Analysis 2000</td><td>Tekno Optic</td><td></td><td></td></tr><tr><td>Streotaxic apparatus</td><td>Stoelting</td><td></td><td></td></tr><tr><td>Graph pad Prism 5</td><td>Graph pad software Inc.</td><td></td><td></td></tr></tbody></table>

protocol for three-dimensional confocal morphometric analysis of astrocytes

如神经胶质细胞在大脑中，星形胶质细胞在中枢神经系统中多样化的功能角色。中的有害刺激的存在下，星形细胞修改他们的功能和结构性质，一个称为反应性星形胶质细胞增生的条件。这里，一个协议的星形胶质细胞的形态性质的评估呈现。这个协议包括12个不同参数，即表面积和所覆盖的星形胶质细胞（星形胶质细胞境）的组织，在整个星形胶质细胞，包括分支，细胞体，和细胞核，体积定量分支以及总长度和数量，强度用于星形胶质细胞的检测抗体的荧光免疫反应性，和星形胶质细胞密度（个/ 1000微米2）。为了这个目的所创建的三维（3D）的共焦显微镜图像和3D图像分析软件如Volocity 6.3用于测量。大鼠脑组织暴露于淀粉样蛋白1-40 </子&gt;（Aβ1-40）具有或不具有治疗性干预是用于呈现方法。该协议也可以被用于其它细胞在体内或体外条件的三维形态测定分析。

本节介绍由三维形态分析产生的定性和定量的观测的一些例子。对于前面所提到的全部12个参数的完整结果，请参考我们以前出版10。 定性观察星形胶质细胞表现出薄或厚的分支中通常是长期的Aβ1-40注射的老鼠（ 图1）。几个小星芒状星形细胞与短支链，观察在大脑皮质，和星形胶质细胞的一种紧凑网络发生在胼胝体。 在动物的脑组织的Aβ1-40 -注射随后染料木黄酮处理的星形胶质细胞表现出与短而细分支的星形形式（图2）10。一些星形胶质细胞表现出萎缩的外观。 星形胶质细胞密度和胶质纤维酸性蛋白+荧光强度星形胶质细胞的平均数目/ 1000微米2是显著高于大鼠Aβ1-40 -注射与在Aβ1-40 -注射的动物和染料木素治疗组 （P &lt;0.0001）比较。此外，GFAP +荧光强度显著降低大脑中的部分与染料木黄酮处理与未处理的Aβ1-40相比注射的动物（图3）。 星形胶质细胞体积形态分析星形胶质细胞细胞核，细胞体，整个星形胶质细胞和星形胶质细胞领土的体积与未处理的动物相比，染料木黄酮治疗组显著下降。这一结果表明，染料木素改善造成的淀粉状蛋白的存在（图4A - D）的所述星形胶质细胞增生。 星形胶质细胞图1.共聚焦图像。一个单独的星形胶质细胞与又粗又长的树枝（箭头）和DAPI标记的细胞核（箭头）在大鼠脑组织受到海马注射Aβ1-40。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53113/53113fig1large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图2" src="/files/ftp_upload/53113/53113fig2.jpg" /> 图中的星形胶质细胞2.共聚焦图像。短分支（箭头）星形胶质细胞。在大鼠脑进行海马Aβ1-40 -注射，和染料木素的前处理通过管饲法给药 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53113/53113fig2large.jpg" target="_blank">。P</a><html>租赁点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53113/53113fig3.jpg" /> 图3. GFAP +荧光强度的动物用Aβ1-40显著下降-注射接受染料木黄酮治疗前值是平均值±标准差。每个动物五十星形胶质细胞进行了评价。P &lt;0.05被认为是显著，以及T检验被用来比较有或无治疗组之间的数据。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53113/53113fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> G"/&gt; 图4.核（A）中，单元主体（B），星形胶质细胞（包括分支的体积平均;从大鼠进行的Aβ1-40 -注射，有或没有采取脑样品（C）中，版图和（D）的金雀异黄素预处理。金雀异黄素显著改善细胞核，细胞体，整个星形胶质细胞和星形胶质细胞的领土（见参考文献10）。值是平均值每头牲畜±SEM。五十星形胶质细胞进行了评价。P &lt;0.05的扩大被视为显著，和T检验用于治疗前后组之间的数据进行比较。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53113/53113fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes at JoVE.com

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

协议星形胶质细胞的三维共聚焦形态分析

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

protocol-for-three-dimensional-confocal-morphometric-analysis

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.8K 次观看.  Linköping University. Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

视频: Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.	
	Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 &mu;m z-axis and a 0.23 &mu;m step size, corresponding to a 180 x 135 x 10 &mu;m volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.

偏振的实验性脑损伤小胶质细胞/巨噬细胞标志物的三维共聚焦分析

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

科研

神经科学

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

生物学

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network level. Astrocytes are strongly connected to each other through gap junctions and coupling through these junctions is dynamic and highly regulated. An emerging concept is that astrocytic functions are specialized and adapted to the functions of the neuronal circuit with which they are associated. Therefore, methods to measure various parameters of astrocytic networks are needed to better describe the rules governing their communication and coupling and to further understand their functions.
Here, using the image analysis software (e.g., ImageJFIJI), we describe a method to analyze confocal images of astrocytic networks revealed by dye-coupling. These methods allow for 1) an automated and unbiased detection of labeled cells, 2) calculation of the size of the network, 3) computation of the preferential orientation of dye spread within the network, and 4) repositioning of the network within the area of interest.
This analysis can be used to characterize astrocytic networks of a particular area, compare networks of different areas associated to different functions, or compare networks obtained under different conditions that have different effects on coupling. These observations may lead to important functional considerations. For instance, we analyze the astrocytic networks of a trigeminal nucleus, where we have previously shown that astrocytic coupling is essential for the ability of neurons to switch their firing patterns from tonic to rhythmic bursting1. By measuring the size, confinement, and preferential orientation of astrocytic networks in this nucleus, we can build hypotheses about functional domains that they circumscribe. Several studies suggest that several other brain areas, including the barrel cortex, lateral superior olive, olfactory glomeruli, and sensory nuclei in the thalamus and visual cortex, to name a few, may benefit from a similar analysis.

Here we present a protocol to assess the organization of astrocytic networks. The described method minimizes bias to provide descriptive measures of these networks such as cell count, size, area, and position within a nucleus. Anisotropy is assessed with a vectorial analysis.

耦合星形胶质细胞网络的大小、形状和方向性分析

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

胶质细胞和神经细胞的三维重建与虚拟现实分析方法

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, which include neurotransmitter clearance, ion regulation, synaptic modulation, metabolic support to neurons, and blood flow regulation. Astrocytes are complex cells that have a soma, several major branches, and numerous fine processes that contact diverse cellular elements within the neuropil. In order to assess the morphology of astrocytes, it is necessary to have a reliable and reproducible method to visualize their structure. We report a reliable&#160;protocol to perform intracellular iontophoresis of astrocytes using fluorescent Lucifer yellow (LY) dye in lightly fixed brain tissue from adult mice. This method has several features that are useful to characterize astrocyte morphology. It allows for three-dimensional reconstruction of individual astrocytes, which is useful to perform morphological analyses on different aspects of their structure. Immunohistochemistry together with LY iontophoresis can also be utilized to understand the interaction of astrocytes with different components of nervous system and to evaluate the expression of proteins within the labelled astrocytes. This protocol can be implemented in a variety of mouse models of CNS disorders to rigorously examine astrocyte morphology with light microscopy. LY iontophoresis provides an experimental approach to evaluate astrocyte structure, especially in the context of injury or disease where these cells are proposed to undergo significant morphological changes.

Astrocytes are morphologically complex cells, exemplified by their multiple processes and bushy territories. To analyze their elaborate morphology, we present a reliable protocol to perform intracellular Lucifer yellow iontophoresis in lightly fixed tissue.

使用路西法黄电磷学可视化星形形态

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

在多地址基因组集成颜色 （MAGIC） 标记的 Utero 电冲中， 以个性化皮质小鼠星形细胞

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

研究清除大脑中星形胶质细胞和神经元之间的空间相互作用

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...

Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the brain. Through these interactions, astrocytes actively regulate many critical brain functions, including synapse formation, neurotransmission, and ion homeostasis. In the rodent brain, astrocytes grow in size and complexity during the first three postnatal weeks and establish distinct, non-overlapping territories to tile the brain. This protocol provides an established method for analyzing astrocyte territory volume and astrocyte tiling using free-floating tissue sections from the mouse brain. First, this protocol describes the steps for tissue collection, cryosectioning, and immunostaining of free-floating tissue sections. Second, this protocol describes image acquisition and analysis of astrocyte territory volume and territory overlap volume, using commercially available image analysis software. Lastly, this manuscript discusses the advantages, important considerations, common pitfalls, and limitations of these methods. This protocol requires brain tissue with sparse or mosaic fluorescent labeling of astrocytes, and is designed to be used with common lab equipment, confocal microscopy, and commercially available image analysis software.

This protocol describes methods for sectioning, staining, and imaging free-floating tissue sections of the mouse brain, followed by a detailed description of the analysis of astrocyte territory volume and astrocyte territory overlap or tiling.

厚自由漂浮组织切片中星形胶质细胞区域体积和瓷砖的分析

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the ...

Immunostaining of Fluorescently Labeled Astrocytes in a Mouse Brain Tissue Section

Source: Eaker, A. R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63804">Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure for immunostaining a fixed mouse brain tissue section with fluorescently labeled astrocytes using primary and secondary antibodies and capture the images of the stained astrocytes through confocal microscopy.

Source: Eaker, A. R., et al. Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections. J. Vis. Exp. (2022)
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协议星形胶质细胞的三维共聚焦形态分析

摘要

探索更多视频

此视频中的章节

偏振的实验性脑损伤小胶质细胞/巨噬细胞标志物的三维共聚焦分析

细胞形态变化，从三维荧光图像量化的分析工具，

耦合星形胶质细胞网络的大小、形状和方向性分析

胶质细胞和神经细胞的三维重建与虚拟现实分析方法

使用路西法黄电磷学可视化星形形态

在多地址基因组集成颜色（MAGIC）标记的 Utero 电冲中，以个性化皮质小鼠星形细胞

研究清除大脑中星形胶质细胞和神经元之间的空间相互作用

厚自由漂浮组织切片中星形胶质细胞区域体积和瓷砖的分析

Immunostaining of Fluorescently Labeled Astrocytes in a Mouse Brain Tissue Section

协议星形胶质细胞的三维共聚焦形态分析

摘要

探索更多视频

此视频中的章节

偏振的实验性脑损伤小胶质细胞/巨噬细胞标志物的三维共聚焦分析

细胞形态变化，从三维荧光图像量化的分析工具，

耦合星形胶质细胞网络的大小、形状和方向性分析

胶质细胞和神经细胞的三维重建与虚拟现实分析方法

使用路西法黄电磷学可视化星形形态

在多地址基因组集成颜色 （MAGIC） 标记的 Utero 电冲中， 以个性化皮质小鼠星形细胞

研究清除大脑中星形胶质细胞和神经元之间的空间相互作用

厚自由漂浮组织切片中星形胶质细胞区域体积和瓷砖的分析

Immunostaining of Fluorescently Labeled Astrocytes in a Mouse Brain Tissue Section

在多地址基因组集成颜色（MAGIC）标记的 Utero 电冲中，以个性化皮质小鼠星形细胞