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>

<ol>
	<li>Siedentopf, H., Uber Zsigmondy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sichtbarmachung+und+Gr&#246;&#223;enbestimmung+ultramikoskopischer+Teilchen,+mit+besonderer+Anwendung+auf+Goldrubingl&#228;ser.">Sichtbarmachung und Gr&#246;&#223;enbestimmung ultramikoskopischer Teilchen, mit besonderer Anwendung auf Goldrubingl&#228;ser.</a> Annalen der Physik. 315 (1), 1-39 (1902).</li><li>Voie, A. H., Burns, D. H., Spelman, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthogonal-plane+fluorescence+optical+sectioning:+three-dimensional+imaging+of+macroscopic+biological+specimens.">Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens.</a> J. Microsc. 170 (Pt 3), 229-236 (1993).</li><li>Epp, J. R., 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=Optimization+of+CLARITY+for+Clearing+Whole-Brain+and+Other+Intact+Organs(1,2,3).">Optimization of CLARITY for Clearing Whole-Brain and Other Intact Organs(1,2,3).</a> eNeuro. 2 (3), (2015).</li><li>Greger, K., Swoger, J., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+building+units+and+properties+of+a+fluorescence+single+plane+illumination+microscope.">Basic building units and properties of a fluorescence single plane illumination microscope.</a> Rev. Sci. Instrum. 78 (2), 023705 (2007).</li><li>Keller, P. J., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+scanned+laser+light+sheet+fluorescence+microscopy.">Digital scanned laser light sheet fluorescence microscopy.</a> Cold Spring Harb Protoc. 2010 (5), (2010).</li><li>Becker, K., Jahrling, N., Kramer, E. R., Schnorrer, F., Dodt, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultramicroscopy:+3D+reconstruction+of+large+microscopical+specimens.">Ultramicroscopy: 3D reconstruction of large microscopical specimens.</a> J Biophotonics. 1 (1), 36-42 (2008).</li><li>Dodt, H. U., 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=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat. Methods. 4 (4), 331-336 (2007).</li><li>Taormina, M. J., 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=Investigating+bacterial-animal+symbioses+with+light+sheet+microscopy.">Investigating bacterial-animal symbioses with light sheet microscopy.</a> Biol. Bull. 223 (1), 7-20 (2012).</li><li>Klingberg, A., 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=Fully+Automated+Evaluation+of+Total+Glomerular+Number+and+Capillary+Tuft+Size+in+Nephritic+Kidneys+Using+Lightsheet+Microscopy.">Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy.</a> J. Am. Soc. Nephrol. , (2016).</li><li>Truong, T. V., Supatto, W., Koos, D. S., Choi, J. M., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+and+fast+live+imaging+with+two-photon+scanned+light-sheet+microscopy.">Deep and fast live imaging with two-photon scanned light-sheet microscopy.</a> Nat. Methods. 8 (9), 757-760 (2011).</li><li>Schmid, B., 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=High-speed+panoramic+light-sheet+microscopy+reveals+global+endodermal+cell+dynamics.">High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics.</a> Nat Commun. 4, 2207 (2013).</li><li>Cella Zanacchi, F., 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=Live-cell+3D+super-resolution+imaging+in+thick+biological+samples.">Live-cell 3D super-resolution imaging in thick biological samples.</a> Nat. Methods. 8 (12), 1047-1049 (2011).</li><li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+Tissue+Clearing.">Clarifying Tissue Clearing.</a> Cell. 162 (2), 246-257 (2015).</li><li>Maruyama, A., 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=Wide+field+intravital+imaging+by+two-photon-excitation+digital-scanned+light-sheet+microscopy+(2p-DSLM)+with+a+high-pulse+energy+laser.">Wide field intravital imaging by two-photon-excitation digital-scanned light-sheet microscopy (2p-DSLM) with a high-pulse energy laser.</a> Biomed Opt Express. 5 (10), 3311-3325 (2014).</li><li>Mann, L., 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=CD11c.DTR+mice+develop+a+fatal+fulminant+myocarditis+after+local+or+systemic+treatment+with+diphtheria+toxin.">CD11c.DTR mice develop a fatal fulminant myocarditis after local or systemic treatment with diphtheria toxin.</a> Eur. J. Immunol. 46 (8), 2028-2042 (2016).</li><li>Jung, S., 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=In+vivo+depletion+of+CD11c++dendritic+cells+abrogates+priming+of+CD8++T+cells+by+exogenous+cell-associated+antigens.">In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens.</a> Immunity. 17 (2), 211-220 (2002).</li><li>Corbi, A. L., Lopez-Rodriguez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD11c+integrin+gene+promoter+activity+during+myeloid+differentiation.">CD11c integrin gene promoter activity during myeloid differentiation.</a> Leuk. Lymphoma. 25 (5-6), 415-425 (1997).</li><li>Zaft, T., Sapoznikov, A., Krauthgamer, R., Littman, D. R., Jung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD11chigh+dendritic+cell+ablation+impairs+lymphopenia-driven+proliferation+of+naive+and+memory+CD8++T+cells.">CD11chigh dendritic cell ablation impairs lymphopenia-driven proliferation of naive and memory CD8+ T cells.</a> J. Immunol. 175 (10), 6428-6435 (2005).</li><li>Zammit, D. J., Cauley, L. S., Pham, Q. M., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+maximize+the+memory+CD8+T+cell+response+to+infection.">Dendritic cells maximize the memory CD8 T cell response to infection.</a> Immunity. 22 (5), 561-570 (2005).</li><li>Hasenberg, M., Kohler, A., Bonifatius, S., Jeron, A., Gunzer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+phagocytosis+and+NET-formation+by+neutrophils+in+infected+lungs+using+2-photon+microscopy.">Direct observation of phagocytosis and NET-formation by neutrophils in infected lungs using 2-photon microscopy.</a> J. Vis. Exp. (52), (2011).</li><li>Bailey, B., Farkas, D. L., Taylor, D. L., Lanni, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+axial+resolution+in+fluorescence+microscopy+by+standing-wave+excitation.">Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation.</a> Nature. 366 (6450), 44-48 (1993).</li><li>Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surpassing+the+lateral+resolution+limit+by+a+factor+of+two+using+structured+illumination+microscopy.">Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.</a> J. Microsc. 198 (Pt 2), 82-87 (2000).</li><li>Hell, S. W., Wichmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+the+diffraction+resolution+limit+by+stimulated+emission:+stimulated-emission-depletion+fluorescence+microscopy.">Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.</a> Opt Lett. 19 (11), 780-782 (1994).</li><li>Betzig, E., 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=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313 (5793), 1642-1645 (2006).</li><li>Hess, S. T., Girirajan, T. P., Mason, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy.">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophys. J. 91 (11), 4258-4272 (2006).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat. Methods. 3 (10), 793-795 (2006).</li><li>Abbe, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beitr&#228;ge+zur+Theorie+des+Mikroskops+und+der+mikroskopischen+Wahrnehmung.">Beitr&#228;ge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung.</a> Archiv f&#252;r mikroskopische Anatomie. 9 (1), 413-418 (1873).</li><li>G&#246;ppert-Mayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#220;ber+Elementarakte+mit+zwei+Quantenspr&#252;ngen.">&#220;ber Elementarakte mit zwei Quantenspr&#252;ngen.</a> Annalen der Physik. 401 (3), 273-294 (1931).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248 (4951), 73-76 (1990).</li><li>M&#228;nn, L., 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=CD11c.DTR+mice+develop+a+fatal+fulminant+myocarditis+after+local+or+systemic+treatment+with+diphtheria+toxin.">CD11c.DTR mice develop a fatal fulminant myocarditis after local or systemic treatment with diphtheria toxin.</a> Eur. J. Immunol. , (2016).</li><li>Ke, M. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+a+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nat. Neurosci. 16 (8), 1154-1161 (2013).</li><li>Erturk, A., 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=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nat. Protoc. 7 (11), 1983-1995 (2012).</li><li>Dirckx, J. J., Kuypers, L. C., Decraemer, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refractive+index+of+tissue+measured+with+confocal+microscopy.">Refractive index of tissue measured with confocal microscopy.</a> J Biomed Opt. 10 (4), 44014 (2005).</li><li>Chung, K., 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497 (7449), 332-337 (2013).</li><li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues.">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nat. Protoc. 9 (7), 1682-1697 (2014).</li><li>Schwarz, M. K., 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=Fluorescent-protein+stabilization+and+high-resolution+imaging+of+cleared,+intact+mouse+brains.">Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains.</a> PLoS One. 10 (5), e0124650 (2015).</li><li>Hama, H., 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=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat. Neurosci. 14 (11), 1481-1488 (2011).</li><li>Susaki, E. A., 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=Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157 (3), 726-739 (2014).</li><li>Yang, B., 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=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158 (4), 945-958 (2014).</li><li>Shivapathasundharam, B., Berti, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+tooth+model+system.+An+aid+in+the+study+of+root+canal+anatomy.">Transparent tooth model system. An aid in the study of root canal anatomy.</a> Indian J. Dent. Res. 11 (3), 89-94 (2000).</li><li>Karreman, M. A., 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=Fast+and+precise+targeting+of+single+tumor+cells+in+vivo+by+multimodal+correlative+microscopy.">Fast and precise targeting of single tumor cells in vivo by multimodal correlative microscopy.</a> J. 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或随机光学重建显微镜（STORM） 26 ，一度假设的分辨率限制光衍射27明显破裂。第二个经常针对性的挑战是在类似环境中观察细胞行为自然条件尽可能接近。在这方面， 原位或体内成像方法的发展被迫。为了克服复杂生物组织中有限的光穿透深度，双光子显微镜28,29为本研究领域树立了新的标准。 然而，所有这些显微技术通常所共有的特征是使用高NA目标来保证最佳分辨率。尽管它更详细地解决了微结构，但是使用高NA目标显着地限制了视野以及工作距离。因此，在周围环境方面的生物环境常常会以最真实的意义而脱离焦点。为了缩小这个差距，LSFM的发展近来有兴趣，提供了一种可以解决细胞大小的荧光结构的显微技术而且还可以方便地评估尺寸超过1厘米的标本的整个3D信息。 这里描述的方案提出了如何使用超显微镜来观察整个小鼠心脏中的细胞白细胞浸润的方法。在CD11c.DTR小鼠模型30中成功诱导无菌心肌炎后，相应的动物接受静脉注射荧光染料偶联的抗CD45抗体，其在活体动物中循环2小时。原则上，通过进行验尸，器官固定和样品脱水之间的全身抗体染色应该是可能的。不幸的是，整体染色需要更长的孵化步骤，因此大大增加了样品生成所需的时间（ 即 4-21天）6,31。此外，这种活体染色方法不仅更快，而且提供抗体与全挂染色方案相比更有效的组织穿透9 。虽然绝对染色效率还没有被量化，但由于所得到的荧光信号非常强，所以预计大部分的白细胞群被标记。荧光蛋白和抗体标记本身在很大程度上仍然存在于随后的清除过程中，并且两者似乎长期稳定。还应注意，溶剂型试剂用于组织清除。在清除过程之后，这大大阻碍了整体染色方法。基于溶剂的组织清除是可逆的现象，所以将清除的组织转移到通常用于标准抗体染色的水基培养基（例如PBS）中将导致不透明的样品。因此，必须建立有机溶剂中的非标准染色方案<html>样品清除后需要全身染色程序。 随后的心脏灌注是绝对必要的，以消除尽可能多的残留血液，因为血液在以下步骤中很差地清除，并且显着地恶化了成像结果。切除心脏后，将组织水以上升的乙醇排除去。这一步可以视为化学清洗的第一部分。这个想法是用浸渍介质来替代组织水，该浸渍介质更好地匹配所选样品的平均折射率（RI）。根据Ertuk 等人的方案，选择DBE作为浸渍介质。 32 ，有一些修改。 DBE的折射率为1.55（nD20），当与其他清除剂如苯甲醇:苯甲酸苄酯（BABB; RI（nD20）= 1.56） 6,7或蔗糖（RI（nD20）= 1.44 ）13，它最好地保存了本协议中使用的荧光标记物，使用肉桂酸乙酯（ECI）产生类似的荧光保存，我们已经描述了它在肾肾肾脏中自动定量肾小球的研究中的应用9。肌肉一次估计折射率为1.382±0.004 33的组织与我们评价的所有物质同样清除9 ，因此，选择DBE的关键因素是荧光保存，而不是清除潜力应该使用这种被动清除方案，使用活性方法如CLARITY 34,35 ，其中借助于电泳电流去除脂质会导致强烈清除的组织，但是，与这样的水相反的清除方法，DBE清除导致显着硬化的组织，伴随轻微收缩。这允许容易地处理样品，并且不需要进一步适应成像，例如将样品包埋在琼脂糖块中。因此，将该协议转移到诸如CLARITY之类的方法将是具有挑战性的，但是人们使用这种方法成功地证明了使用所选择的超显微镜（参见材料表）。重要的是通过选择的清除方法仔细评估内源性荧光信号的潜在破坏。由于我们从未与CLARITY或其他主动清除协议一起使用，所以我们不能评论这些系统中的荧光染料反应。最坏的情况是必须执行随后的全抗体染色。 该方法易于调节其他器官，如鼠肺;肾脏;大脑;甚至骨头，如股骨，胫骨或颅盖。评价时<html>在新组织区域和/或新的荧光标记策略上应用潜力，最大的挑战通常是以荧光保持活性的方式设计方案。溶剂型浸渍介质13和脱水剂36对荧光染料的完整性具有直接影响。推荐使用AlexaFluor衍生物，因为它们在各种方案中似乎相当稳定。然而，通常必须使用其它荧光染料。在这种情况下，绝对值得评估其他清除剂的使用（ 例如， Scale 37 ，Cubic 38 ，Succrose 13 ，Clarity 13,34，PACT 39 ，FocusClear 13 ，SeeDB 31 ，水杨酸甲酯40，3Disco 32 ，BABBlass ="x ref"&gt; 6和ECI 9 ）和其他脱水替代品（ 如甲醇和四氢呋喃13 ）。 像许多其他的LSFM方法一样，透明心脏在显微镜下的定位过程是非常重要的。商业系统提供足够的空间来安装小鼠器官（包括肺叶，肾脏或心脏）的大小的样本。但是，目前还没有标准样品架可供选择。因此，我们必须通过使用清除和重新形状的琼脂糖块来建立安装设备。然而，由于运动伪影，一定数量的图像数据集必须被丢弃。由于这里使用的显微镜​​的长采集时间，这个问题也增加了。潜在地，巨大的数据集的产生将从更快的系统中获益匪浅，但是我们没有机会使用更快的显微镜。我们的显微镜（见表of材料）围绕变焦显微镜体构建，配备有激光模块，像素尺寸为6.5×6.5mm 2的sCMOS照相机，以及光学倍率范围为1.26X至12.6X的检测光学器件。检测光学元件的中心部分为0.5 NA物镜，工作距离为5.5 mm，可观察到对角线范围在1.7至17.6 mm之间的大视场。这足够大，可以进行整个小鼠心脏的光学切片。 400至800 nm之间的色差校正是通过物镜的机械调整实现的。在需要时，在计算机辅助后期处理中进一步进行色彩校正。在该系统中，没有观察到球面像差，也是较厚的样品，因此不需要校正。 总而言之，我们在这里介绍一种用于空间l的化学清除和分析的可靠协议使用光照片显微镜在小鼠心脏内部的白细胞分布。在这一点上，重要的是要强调，这种LSFM程序很难解决诸如毛细血管的细小组织结构。在这方面，这种LSFM非常适合表征一般的免疫细胞分布和描述器官内潜在的免疫细胞积累。然而，必须牢记，LSFM一般不是分析和量化小器官亚结构（如脉管系统）细胞定位的完美选择。为了回答这样的问题，LSFM应该补充其他方法，如组织学或双光子显微镜。该协议的进一步限制包括限制到最多3-4种颜色 - 蓝色通道难以用于除自发荧光之外的其他信号 - 对固定样品的限制，最大样品尺寸为30 mm x 30 mm x 15毫米。此外，应采取DBE的强烈的气味和急性毒性考虑到。如果后一点存在问题，可以使用肉桂酸乙酯作为替代清除剂9 。选择这种显微技术的主要优点是能够一次分析整个器官。在苏木精和伊红染色，福尔马林固定和石蜡包埋（FFPE）组织切片的常规分析中，除非分析每一个部分，否则很可能缺少重要的空间区域。此外，光照显微镜不仅可以在每个三维平面上虚拟切割样品，而且还可以在不破坏样品或引起切割伪影的情况下提供此潜力。如果需要，固定器官可以随后进行进一步分析处理，例如相关的光和电子显微镜方法以高度解决感兴趣的特定区域。 Karreman 等人的工作中可以找到这样一个研究的模板。 ，其中相关赌注弱化双光子显微镜图像堆叠和三维电子显微镜数据成功显示41 。另一个有希望的方法可以是该方案与最近公布的uDISCO方法的组合，该方法也基于有机溶剂，并且将允许通过标准LSFM 42对更大的器官进行调查。

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液。如果成功执行，心脏和其他器官发白。随后，切除器官，组织水通过上升的乙醇排和DBE中的最终孵育与DBE交换。 图1B显示了时间表的器官经历显着收缩的个体孵育步骤。然后将化学清除的器官转移到标准显微镜组织支架上的DBE填充的成像室中。为了在3D图像采集期间稳定器官，它由透明琼脂糖块固定。样品的详细定位可见于图2 。由2个通道（自动荧光和抗CD45）组成的整个图像堆栈的获取大约需要20-30分钟，并生成大约16 GB的数据文件。最后，将所得数据分析为人造3D渲染，其中白细胞分布在热图中显示。在图3 （上排）中，DTX处理的​​动物的心脏的强烈发炎的区域可以被其红色/发白的外观感觉到。对3D模型的更详细的研究揭示了白细胞浓度，特别是在心脏传导系统的区域，例如动脉粥样硬化<html>针状束和浦肯野纤维。相比之下，PBS处理的动物的心脏根本不显示这种炎症模式（下排）。 <img alt="图1" src="/files/ftp_upload/55450/55450fig1.jpg" /> 图1:白喉毒素诱导的心肌炎的诱导和样品制备方案。 （ 一 ）心肌炎的发生是由它引起的。应用白喉毒素（100ng /只）。 4天后，心肌炎充分发育，注射AF647偶联的抗CD45抗体（15μg/只） 。检测CD45 +白细胞。 2小时后通过颈脱位处死动物，并用PBS / EDTA（5mM），随后4％PFA灌注心脏。 （ B ）去除后，将器官用上升的乙醇系列脱水，最后通过在二苄基醚中过夜孵育而清除。.com / files / ftp_upload / 55450 / 55450fig1large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图2" src="/files/ftp_upload/55450/55450fig2.jpg" /> 图2:显微镜样品定位方案。化学清除的透明心脏（4）定位在样品架（2和5）上，并借助于清除的琼脂糖块（6）稳定。然后将该构造物浸没在装有DBE的玻璃比杯（3）中。对于采集，使用工作距离为5.5mm的0.5NA物镜（1）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55450/55450fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55450/55450fig3.jpg" /> 图3:全代表的3D渲染代表gan光照显微镜。 CD11c.DTR小鼠用DTX（上排）或PBS（下排）进行气管内治疗。 4天后，通过荧光光谱显微镜观察由CD45 +白细胞浸润代表的心肌炎的发展。为了染色CD45 +细胞，在处死小鼠前静脉内注射与AF647偶联的抗CD45抗体，静脉注射2小时。结缔组织的自发荧光信号显示为灰色，而CD45 +浸润显示为热图颜色（蓝色:低信号;白色:高信号）。每行由4个数字剪辑的3D渲染组成，显示器官内的情况。相对于心脏前部的剪切深度被声明在单个图像之上。该图已从Mann 等人修改15 。比例尺= 1 mm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55450/55450fig3large.jpg" target="_blank">请点击这里</a>以查看该图的较大版本。

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

Nanyang Technological University

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

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 ...

科研

免疫与感染

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 ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

使用真空稳定成像系统的实验性急性肺损伤中肺微循环的活体内宽视场荧光显微镜

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

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

探索更多视频

此视频中的章节

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

使用真空稳定成像系统的实验性急性肺损伤中肺微循环的活体内宽视场荧光显微镜

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

使用真空稳定成像系统的实验性急性肺损伤中肺微循环的活体内宽视场荧光显微镜

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