Name: quantitative visualization of leukocyte infiltrate in a murine model of fulminant myocarditis by light sheet microscopy
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Description: Watch this Scientific Journal Video about Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy at JoVE.com

Gunzer实验室的研究得到德国联邦教育和研究部（公元0315590）的授权和德国研究基金会（授权号GU769 / 4-1，GU769 / 4-2）的支持。我们进一步感谢IMCES的技术支持和Sebastian Kubat对3D卡通造型的帮助。

所有动物实验均按照欧盟指南进行，并得到了埃森相关地方当局的批准（AZ 84-02.04.2014.A036 - LandesamtfürNatur，Umwelt und Verbraucherschutz Nordrhein-Westfalen，Essen，Germany）。使用动物并在特定无病原体（SPF）条件下饲养。 白喉毒素诱导心肌炎（DTX） <ol><li>通过将DTX储备溶液用磷酸盐缓冲盐水（PBS）稀释到1μg/ mL工作溶液中，制备DTX溶液用于诱导心肌炎。 </li><li> 应用100μL这种溶液气管内（它）到8至10周龄的CD11c.DTR小鼠（ 约 5ng / g体重（bw）），如Hasenberg 等人所示。为了应用真菌孢子悬浮液20 。 注意:腹膜内施用毒素可能导致类似的诱导致命性心肌炎。但是，这种方法必须首先被验证。 <ol><li>对于它的应用，通过腹膜内（ip）注射100mg / kg bw氯胺酮和10mg / kg bw赛拉嗪麻醉动物。 </li><li>达到深度麻醉（脚趾压缩测试）后，通过使用22 G静脉导管通过口腔插管动物。通过使用小型动物呼吸器以每分钟250次呼吸和每次呼吸250μL的吸入体积通气，检查镜片管的正确定位，Hasenberg 等也显示。人。 20 。 注意:正确定位时，动物的呼吸速率将根据应用的通气设置而改变。 </li><li>断开通气系统，并将100μLDTX溶液或等量的PBS作为对照通过导管滴入肺内，并继续对动物进行再通气1分钟。 </li></ol></li><li>让动物从麻醉中恢复，并监测一般健康状况和体重变化4天。 注意:根据遗传背景，小鼠应变或初始体重，心肌炎的严重程度和发作可能有所不同，应首先确定。 </li></ol> 2.光照显微镜的样品制备<ol><li>毒素施用后4天，用蒸发的2％异氟烷/氧气混合物冲洗诱导室，麻醉小鼠。使用后视镜应用途径静脉内（iv），在50μLPBS中注射15μg直接标记的抗CD45抗体（克隆30-F11，AlexaFluor647（AF647）），这确保了非常快速和高度准确的应用正确量的抗体。 注意:当小鼠躺卧并且不对脚趾夹紧测试做出反应时，达到了所需的麻醉深度。 </li><li> 孵育2小时后，通过颈椎脱位处死小鼠d用5 mL PBS / EDTA（5 mM）灌注心脏。 <ol><li>使用21 G导管将心脏暴露于右心室注射灌注溶液。灌注缓慢，恒定的压力，并确保在切开主动脉后可以排出血液。 注意:这种经心脏灌注可能会导致较小的准备工件，这可能会干扰器官的最终3D可视化。因此，先进的动物实验者可以通过下腔静脉进行灌注，切除腹主动脉。 </li></ol></li><li> 对于化学固定，通过与5mL的4％多聚甲醛（PFA）相同的凹处灌注心脏。将导管保持在适当位置，然后轻轻地安装一个装有PFA的新注射器。充满缓慢和恒定的压力。 注意:对甲醛有剧毒性;避免与皮肤，眼睛和其他粘膜接触。 <ol><li>用锯齿镊子轻轻抓住心脏; PU稍微向上;并切割所有组织连接，包括进入和输出的动脉和静脉。将器官在4％PFA中另存4 h，以进一步固定。 </li></ol></li><li>为了使器官脱水，以50％，70％和100％的上升乙醇系列将心脏孵育12小时，在黑暗中4℃孵育。在这里，使用黑色的5毫升聚丙烯反应管，使其能够以较慢的转速在管旋转器中恒定旋转，以防止气泡形成。 </li><li>通过化学清除完成样品制备。为了使心脏透明，将其在98％二苄醚（DBE）中孵育，同时不断旋转过夜。 注意:DBE刺激眼睛，皮肤和呼吸系统。 </li></ol> 3.光照显微镜<ol><li>使用光照显微镜进行LSFM（参见材料表）。将样品放在显微镜的标准样品架上放入DBE填充的铜中vette，适用于高达30 mm x 30 mm x 15 mm的样品。 </li><li> 在使用脱水和清除的琼脂糖块的图像采集过程中将样品牢牢固定在适当位置。 <ol><li>为了制备琼脂糖块，将2％熔融琼脂糖倒入模具（15 mm x 15 mm x 5 mm）。让琼脂糖冷却下来，直到凝固。 </li><li>以升序乙醇系列（50％，70％，100％100％）脱水。在98％DBE中孵育琼脂糖块过夜。将琼脂糖储存在DBE中直至使用。 注意:由于琼脂糖主要由水组成，每个脱水步骤的孵育时间可以根据块的大小而延长。 注意:DBE刺激眼睛，皮肤和呼吸系统。 </li><li>当需要时，使用锋利的手术刀将制备的琼脂糖块切割成所需的形状。通常，切割棱镜或楔形形状以放置在样品适配器的边缘。 注意:由于琼脂糖块具有弹性，当用力稍微推动时，样品保持原位。 </li></ol></li><li> 用适当的激发和发射滤光片设置检测感兴趣的荧光信号（这里为自发荧光和抗CD45抗体染色）。 <ol><li>为了检测结缔组织来源的自发荧光信号，使用波长488 nm（OPSL:50 mW）的525/50 nm带通滤波器。在668nm（带通滤波器:680/30nm）和647nm的激发波长（二极管激光器:50mW）下检测到具有AF647偶联抗体的CD45染色。 </li><li>选择4μm作为两个单独z平面之间的距离。 </li></ol></li></ol>图像后处理注意:采用科学的3D / 4D图像处理和分析软件进一步处理获取的数字图像数据（参见材料表）。 <ol><li> 打开和预先调整数据文件。 </st荣&gt; <ol><li>开始分析软件后，选择左上角的"Surpass"按钮。要打开图像堆栈，请选择"文件"，"打开"，然后选择包含第一个获取的通道数据的文件夹。选择"编辑"和"添加频道"以添加更多获取的频道。 注意:根据数据大小和计算机系统，此过程可能需要几分钟。所呈现的协议演示了2个采集通道（自发荧光和AF647）的所有步骤。 </li><li>通过单击"编辑"并选择"图像属性"来更正x，y和z体素尺寸。当新窗口打开时，调整参数。 注意:此步骤取决于所用的显微镜​​系统和特定的数据格式。正确的xyz值对于随后的尺寸和远距离测量是至关重要的。在大多数情况下，这些参数作为元数据存储在显微图像文件中。但是，如果文件fo分析软件无法正确解释rmat，手动输入像素大小很重要。 x和y尺寸中的像素尺寸取决于相机像素大小，潜在应用的合并，以及镜头和物镜放大倍数。 z中的像素大小等于自定义的z步长（ 例如 4μm）。 </li></ol></li><li> 频道调整 <ol><li>首先通过打开"显示调整"（"编辑"和"显示调整"）将自动荧光信号转换为灰度。新窗口将列出所有打开的通道和所有显示设置;要更改其默认颜色，选择自动荧光通道并选择"白色"显示样式。点击"确定"以应用。 </li><li>对于黑色电平调整，返回"显示调整"，并调整黑色电平值，以排除不代表样品结构的不需要的背景信号秒。 <ol><li>为此，请使用通道栏上的左上角三角形并将其从左向右拖动。 注意:更改将立即可视化。确保仅抑制不是从样品得到的信号。背景中的最小值不应该是"0"，以避免音调黑色像素，因为可能需要噪声信号进行额外的计算。确保在图像采集后执行所有黑色电平变化。否则，宝贵的样品信息可能会丢失。黑色调整只是代表性的目的。要在不同的样品上使用相同的显示设置，可以在通道列表下面的"最小"数值区域中输入精确的值。这对定量分析非常有帮助。 </li></ol></li><li>执行强度调整，要么通过使用右上角的三角形来调整相应的通道强度，要么将精确值输入到通道下方的"最大"数值范围列表。 </li><li>通过拖动通道条中间的下三角形来进行对比度调整，以增加或减少伽马值或输入精确值。 </li><li>根据自动荧光通道调整AF647通道。不同的是，将AF647信号的可视化设置为热图颜色查找表。通过类似于步骤4.2.1的方法选择频道模式"火"。 注意:由于整体信号强度与自发荧光通道相比显着降低，黑色电平通常调整到更低的值。对于自发荧光通道，该通道的亮度通常增加。 </li><li>选择"混合模式"来细化组织结构。用相同的参数值分析来自一个系列的所有样品。 </li></ol></li><li> 虚拟剪辑 <ol><li>在3D场景展示之前虚拟切割器官rt，在渲染的3D对象上应用裁剪平面。 <ol><li>点击对象列表中的"剪切平面"图标（剪刀）。在图像视图中，将出现黄色框架和操纵器（白色主轴）。用鼠标旋转较薄端的主轴，从而改变裁剪平面的方向，并移动主轴较厚的中间部分，以选择所需的裁剪深度。 </li><li>通过取消选中对象列表下方的相应复选框来隐藏框架和操纵器，并创建所需的快照。 </li></ol></li></ol></li></ol>

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Cell Sci. 129 (2), 444-456 (2016).</li><li>Pan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shrinkage-mediated+imaging+of+entire+organs+and+organisms+using+uDISCO.">Shrinkage-mediated imaging of entire organs and organisms using uDISCO.</a> Nat. Methods. 13 (10), 859-867 (2016).</li></ol>

作者没有什么可以披露的。

在现代生命科学中，生物过程的微观可视化起着越来越重要的作用。在这种情况下，在过去两个世纪里已经取得了许多发展，帮助回答到目前为止无法解决的问题。首先，从根本上提高光学显微镜的分辨能力有明显的趋势。使用结构照明显微镜（SIM） 21,22 ，受激发射耗尽（STED）显微镜23和光激活定位显微镜（PALM） 24,25或随机光学重建...

在光学显微镜的进化过程中，出现了许多专门的形式，它们都被开发出来，以最小化特定标本的可视化过程的限制。一种这样的方法是光片荧光显微镜（LSFM）。首先在1903年由Siedentopf和Zsigmondy 1开发 ，并在20世纪90年代早期找到了其第一个基本的生物应用2 ，LSFM已经成为迄今为止最大的微观工具，可以显示大样本，如完整的小鼠器官，具有荧光信号分辨率下降到细胞水平。由于这些优点，结合其活细胞成像的潜力，自然方法将LSFM命名为"2014年度方法3 "。 顾名思义，LSFM中的样品照明由光片进行，光片垂直于所用物镜的轴定向或发射光收集和随后的图像形成。光板通常通过将宽的准直激光束与柱面透镜聚焦或者通过在仅一个水平或垂直平面4,5中的窄的聚焦激光束的快速侧向运动来产生。以这种方式，只有成像光学器件的焦平面被照亮，通常具有1-4μm的厚度。因此，对于放置在照明平面中的荧光样品，消除或大大抑制6,7的散射光的产生和来自焦平面上方或下方的区域的光漂白的影响。由于所有失焦的飞机都不被照亮，所以在这些区域中不会出现光漂白效果。与标准共聚焦或多光子显微镜相反，照明和发射光的路径彼此分离，因此最终图像质量<html>不依赖于通过目标的激发光束的完美聚焦。根据潜在的问题，因此可以使用具有巨大视场（FOV）的物镜，使得照射平面的最大可能面积可被成像，而没有任何部件在xy方向上移动。 在现代LSFM系统中，产生的光学部分的荧光图像被捕获在能够获得整个视场（FOV）的高灵敏度电荷耦合器件（CCD）或互补金属氧化物半导体（CMOS）照相机上，微秒。因此，通过将样品移动通过光片并通过以确定的z步骤获取图像，可以在合理的时间量8,9获得样本的完整3D信息，使得该技术适用于活细胞研究10 ，ass ="xref"&gt; 11，12。 然而，尽管LSFM提供了一种快速，灵敏和荧光友好的方法，但通过样品的透光性仍然是一个主要问题，特别是当大型生物样品成为3D分析的目标时。通过吸收的物理方面和具有不同折射率的结构的界面处的光散射来严格地改变透光性。因此，当大小成像采样数毫米时，LSFM主要与清除协议相结合，使样本光学透明。这些技术基于从相应的生物组织中除去水并将其与基于溶剂的浸渍介质（基于溶剂的浸渍介质）进行交换的想法，所述浸渍介质被选择为与特定目标组织组分的折射率窄度匹配。结果，横向光散射最小化，并且所有的波长的光可以更有效地穿过组织13 。在许多情况下，以这种方式处理的生物标本在宏观上看起来是晶体透明的，即使在整个小鼠器官上，使用长工作距离，低放大倍数的目标，也可以进行LSFM。 在这里，我们提出了在光照片显微镜（参见材料表）中大样本成像的准备方案，我们已经建立了这一方案来研究心肌炎鼠模型中的细胞心脏浸润15 。 CD11c.DTR小鼠在CD11c启动子16的控制下表达灵长类白喉毒素受体（DTR）。因此，与DTR一起表达CD11c的这些小鼠中的细胞对白喉白喉毒素（DTH）的外毒素敏感 ;用DTX对这些动物的系统治疗导致所有CD11c + ce的耗尽LLS。 CD11c是整联蛋白，并且作为各种不同可溶性因子的细胞表面受体，主要参与单核细胞谱系17的细胞的激活和成熟过程。因此，CD11c.DTR小鼠模型已被广泛用于研究树突状细胞和巨噬细胞亚群在许多不同免疫学问题背景下的作用。随着时间的推移，据报道，用DTX系统治疗的CD11c.DTR小鼠可能会​​产生不良副作用，并可显示强烈升高的死亡率18,19 。最近，我们能够确定死因的原因15 ，描述了在这些动物中气管内DTX应用后暴发性心肌炎的发展。毒素挑战引起细胞破坏，包括在心脏刺激传播系统的中心部分。伴随着大量的CD45+白细胞浸润，最终导致致命的心律失常。在这种情况下，不仅白细胞群体的出现是重要的，而且也是其心脏内部的空间分布。这个实验问题是现代显微成像的一个挑战，我们已经通过光镜显微镜方法解决了这一点，该方法由活体抗体染色和基于有机溶剂的光学清除方案支持。

<table><tbody><tr><td>diphtheria toxin</td><td>Sigma Aldrich</td><td>D0564</td><td></td></tr><tr><td>phosphate buffered saline</td><td>Biochrom</td><td>L182-10</td><td></td></tr><tr><td>ketamine [50 mg/mL]</td><td>Inresa</td><td>PZN 4089014</td><td></td></tr><tr><td>xylazine [2 %]</td><td>Ceva</td><td></td><td></td></tr><tr><td>syringe</td><td>Braun</td><td>REF 9161502</td><td>Braun Omincan F&amp;nbsp;</td></tr><tr><td>indwelt venous catheter&amp;nbsp;</td><td>Becton Dickinson</td><td>REF 381923</td><td>BD Insyte Autoguard</td></tr><tr><td>small animal respirator</td><td>Harvard Apparatus</td><td>73-0044</td><td>MiniVent</td></tr><tr><td>anit-CD45 antibody (AF647)</td><td>BioLegend</td><td>103124</td><td></td></tr><tr><td>ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate</td><td>Carl Roth</td><td>8043.2</td><td></td></tr><tr><td>catheter (21 G)</td><td>BD Biosciences</td><td>REF 387455</td><td>BD valu-set</td></tr><tr><td>paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td></td></tr><tr><td>PE tube (5 mL)</td><td>Carl Roth</td><td>EKY9.1</td><td>Rotilabo</td></tr><tr><td>ethanol</td><td>Carl Roth</td><td>9065.4</td><td></td></tr><tr><td>dibenzyl ether</td><td>Sigma-Aldrich</td><td>108014</td><td></td></tr><tr><td>tube rotator</td><td>Miltenyi Biotec</td><td>130-090-753</td><td>MACSmix</td></tr><tr><td>agarose (low gelling)</td><td>Sigma Aldrich</td><td>A4018</td><td></td></tr><tr><td>mold (15 x 15 x 5 mm)</td><td>Tissue Tek</td><td>4566</td><td>Cryomold&amp;nbsp;</td></tr><tr><td>light sheet microscope system</td><td>LaVision Biotec</td><td></td><td>Ultramicroscope&amp;nbsp;</td></tr><tr><td>microscope body</td><td>Olympus</td><td></td><td>MVX10&amp;nbsp;</td></tr><tr><td>objective</td><td>Olympus</td><td></td><td>0.5&amp;nbsp;NA MVPLAPO 2XC, WD 5.5&amp;nbsp;mm&amp;nbsp;</td></tr><tr><td>sCMOS camera 5.5MP</td><td>Andor Technology</td><td></td><td></td></tr><tr><td>488 nm OPSL (50mW) laser</td><td>Coherent</td><td></td><td></td></tr><tr><td>647 nm&amp;nbsp; diode laser (50mW)</td><td>Coherent</td><td></td><td></td></tr><tr><td>3D image processing &amp;amp; analysis software</td><td>Bitplane</td><td></td><td>IMARIS Ver. 8.3</td></tr><tr><td>transgenic mouse strain</td><td>Steffen Jung et al.</td><td></td><td>CD11c.DTR</td></tr><tr><td>wt mouse strain</td><td>Envigo</td><td></td><td>Balb/c Ola Hsd J</td></tr><tr><td>Laser Module</td><td>LaVision Biotec</td><td></td><td></td></tr><tr><td>MVPLAPO 2XC Objective Lens</td><td></td><td></td><td></td></tr></tbody></table>

quantitative visualization of leukocyte infiltrate in a murine model of fulminant myocarditis by light sheet microscopy

光谱荧光显微镜（LSFM）与化学清除方案相结合，已经成为分析大型生物标本中荧光标记结构的黄金标准，并且归功于细胞分辨率。同时，不断改进的底层协议和增强的专业商业系统的可用性使我们能够调查整个小鼠器官的微结构，甚至允许表征各种活细胞成像方法中的细胞行为。在这里，我们描述了空间整体可视化和发射小鼠心脏CD45 +白细胞群体定量的方案。该方法采用最近已被证明可用作研究暴发性致命性心肌炎发展的鲁棒诱导型模型的转基因小鼠株（CD11c.DTR），其特征在于致死性心律失常。该方案包括心肌炎诱导，玻璃体内抗体介导的细胞染色，器官制备和随后的计算机辅助图像后处理的LSFM。尽管该方案作为我们特殊科学问题的高度适应性方法，但协议代表了易于调整的系统的蓝图，也可以在其他器官甚至其他物种中完全不同的荧光结构。

所提出的LSFM方法分析诱导重度心肌炎时小鼠心脏中的白细胞分布和量。 图1A说明转基因CD11c.DTR小鼠心肌炎诱导的预处理方案。该步骤代表将白细胞募集到心肌的必要触发因素。 DTX应用成功后，动物会发生严重的疾病症状，如2-4天的全身无力，厌食，体重减轻。 4天后，白细胞被iv染色。在经心脏灌注之前应用荧光染料偶联的抗CD45抗体以从循环中除去血?...

Watch this Scientific Journal Video about Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy at JoVE.com

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

在这里，我们描述了在无菌暴发性心肌炎的鼠模型中可视化心脏CD45 +白细胞浸润的光照片显微镜方法，其由气管内白喉毒素治疗CD11c.DTR小鼠诱导。

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

quantitative-visualization-leukocyte-infiltrate-murine-model

Research

JoVE Journal

Medicine

7.7K 次观看.  University of Duisburg/Essen. 在这里，我们描述了在无菌暴发性心肌炎的鼠模型中可视化心脏CD45 +白细胞浸润的光照片显微镜方法，其由气管内白喉毒素治疗CD11c.DTR小鼠诱导。 

视频: Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

科研

免疫与感染

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

白细胞亚群在暂时或永久的脑缺血模型体视学和流式细胞仪鉴定

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

医学

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

光片荧光显微术在小鼠心脏研究中的应用

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

生物工程

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

通过体内显微镜评估的小鼠中白细胞肌渗透

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease&#39;s complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

The present protocol describes light-sheet fluorescent microscopy and automated software-assisted methods to visualize and precisely quantify proliferating and dormant Trypanosoma cruzi parasites and T cells in intact, cleared organs and tissues. These techniques provide a reliable way to evaluate treatment outcomes and offer new insights into parasite-host interactions.

完整澄清器官中克 氏锥虫感染细胞、休眠无鞭毛虫和 T 细胞的定量 3D 成像

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi ...

用于全面心脏成像的光片显微镜和组织透明化

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights into fundamental cardiac physiology and pathologic responses. We hereby delve into the development and implementation of the LSM technique to elucidate the micro-architecture of the heart in mouse models. The methodology integrates a customized LSM system with tissue clearing techniques, mitigating light scattering within cardiac tissues for volumetric imaging. The combination of conventional LSM with image stitching and multiview deconvolution approaches allows for the capture of the entire heart. To address the inherent trade-off between axial resolution and field of view (FOV), we further introduce an axially swept light-sheet microscopy (ASLM) method to minimize out-of-focus light and uniformly illuminate the heart across the propagation direction. In the meanwhile, tissue clearing methods such as iDISCO enhance light penetration, facilitating the visualization of deep structures and ensuring a comprehensive examination of the myocardium throughout the entire heart. The combination of the proposed LSM and tissue clearing methods presents a promising platform for researchers in resolving cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

作者聚焦：用于研究啮齿动物心脏心肌结构的定制光片成像

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

光片成像揭示啮齿动物心脏的心脏结构

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights ...

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

摘要

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此视频中的章节

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

白细胞亚群在暂时或永久的脑缺血模型体视学和流式细胞仪鉴定

光片荧光显微术在小鼠心脏研究中的应用

通过体内显微镜评估的小鼠中白细胞肌渗透

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

完整澄清器官中克氏锥虫感染细胞、休眠无鞭毛虫和 T 细胞的定量 3D 成像

光片成像揭示啮齿动物心脏的心脏结构

光片成像揭示啮齿动物心脏的心脏结构

光片成像揭示啮齿动物心脏的心脏结构

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

摘要

探索更多视频

此视频中的章节

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

白细胞亚群在暂时或永久的脑缺血模型体视学和流式细胞仪鉴定

光片荧光显微术在小鼠心脏研究中的应用

通过体内显微镜评估的小鼠中白细胞肌渗透

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

完整澄清器官中克 氏锥虫感染细胞、休眠无鞭毛虫和 T 细胞的定量 3D 成像

光片成像揭示啮齿动物心脏的心脏结构

光片成像揭示啮齿动物心脏的心脏结构

光片成像揭示啮齿动物心脏的心脏结构

完整澄清器官中克氏锥虫感染细胞、休眠无鞭毛虫和 T 细胞的定量 3D 成像