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?...

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
		</tr>
		<tr>
			<td>indwelt venous catheter&#160;</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 (15x15x5 mm)</td>
			<td>Tissue Tek</td>
			<td>4566</td>
			<td>Cryomold&#160;</td>
		</tr>
		<tr>
			<td>light sheet microscope system</td>
			<td>LaVision Biotec</td>
			<td></td>
			<td>Ultramicroscope&#160;</td>
		</tr>
		<tr>
			<td>microscope body</td>
			<td>Olympus</td>
			<td></td>
			<td>MVX10&#160;</td>
		</tr>
		<tr>
			<td>objective</td>
			<td>Olympus</td>
			<td></td>
			<td>0.5&#160;NA MVPLAPO 2XC, WD 5.5&#160;mm&#160;</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&#160; diode laser (50mW)</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D image processing &#38; 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 ...

Research

JoVE Journal

Medicine

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

一个小鼠模型，通过神经肌肉电刺激肌肉训练

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

科研

医学

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Murine Model of Wound Healing

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

A Murine Model of Subarachnoid Hemorrhage

蛛网膜下腔出血的小鼠模型

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

可重放截瘫胸主动脉阻断脊髓缺血再灌注的小鼠模型

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

心脏异常的实验性自身免疫性心肌炎的无创性评估磁共振成像显微镜在鼠标

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening

CoCrMo 颗粒暴露的小鼠颅骨骨溶解模型的构建与评价

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long ...