这项研究是由INSERM，艾克斯 - 马赛大学和HFSPO由CL弗赖斯获得职业发展奖的支持。

伦理声明：所有的动物研究是按照批准了埃克斯 - 马赛UNIVERSITE，法国的机构动物护理和使用委员会的指导方针和协议执行。雌性C57BL / 6小鼠在8 - 10周，根据DécretN°8 87-848，1987年10月19日，巴黎的规则购得和处理。全部采用L.实验原虫 LD1S寄生虫根据来自法国和欧洲联盟立法生物安全法规进行。 1.鼠标感染L.原虫鞭毛体寄生虫<ol><li>制备培养基进行体外维持L的原虫鞭毛体寄生虫，LD1S（MHOM / SD / 62 / 1S-CL2D）。 <ol><li>使用M199中。补充M199培养基，用10％胎牛血清（加热灭活在56℃，30分钟），25毫米的HEPES pH值6.9，12mM的碳酸氢钠 ，1mM的谷氨酸tamine，RPMI 16040维生素混合物，10毫叶酸，100mM的腺苷，7.6毫血红素，50U / ml青霉素和50mg / ml链霉素。 </li></ol></li><li>培养LD1S寄生虫在10ml完全M199培养基的在26℃在25 cm 2的通风烧瓶中。 </li><li>连续使用差速离心步骤，准备一个富含亚循环前鞭毛体形式的寄生虫人口。 <ol><li>收获了数中期寄生虫文化。指望一个hemocytomer。重悬5×10 5个寄生虫/ ml的在10ml完整的M199培养基中培养。生长的寄生虫培养在厌氧条件下，直到它们到达晚固定相（在约5-6天）。 </li><li>旋LD1S培养于1200×g离心10分钟，在26℃，以沉淀非亚循环寄生虫，回收上清液，并在2,500 xg离心再次旋转它在26℃下进行10分钟。 注意：此最终沉淀高度富集亚循环前鞭毛体寄生虫。 </li><li>悬浮在1颗粒00微升PBS。删除一个小等份固定用25％戊二醛1/100体积：体积和使用血球计数的寄生虫。 </li></ol></li><li>稀释富集-亚循环寄生虫种群至1×10 一百分之七微升PBS中。注入寄生虫悬浮到尾静脉麻醉小鼠的（见下文），使用1ml注射器与地下30针。对于对照动物，注入100μl的PBS中至尾静脉。 </li></ol> 2.外科手术<ol><li>消毒用杀生物喷雾消毒的工作空间和所有的手术器械用70％乙醇30分钟。 </li><li>制备的麻醉剂溶液，根据动物的体重，含有氯胺酮125毫克/公斤和12.5毫克/千克甲苯噻嗪的在PBS中稀释。 </li><li>称重小鼠并通过腹膜内用1ml注射器与地下30针注射麻醉剂的适当剂量。 </li><li>按住鼠标温暖和检查鼠标是完全anesthe通过继续之前捏垫脚级分配。重新注入在小鼠每隔60分钟的麻醉剂溶液的一半剂量。 </li><li>刮胡子鼠标的腹部，并与Vetedine清洁。使一个小切口在腹部的左侧胸廓下皮肤和肌肉。 </li><li>躺在刚打开面积腹部下方的一块湿纱布。暴露肝脏，将它放在纱布。躺在另一块纱布湿润肝上的。 </li><li>放置一个24×60毫米2到150微米厚的盖玻片，氰基丙烯酸酯，结合到金属框架，在显微镜下肝脏进行可视化。 </li><li>保护的小鼠的眼中，用湿纱布。 </li><li>成像会话后，安乐死颈椎脱位的麻醉动物。 </li></ol>肝脏结构的活体3显微成像<ol><li>开展活体显微镜实验上的倒置共聚焦显微镜配备有hermostatic受控室，复消色差63X油甘油浸没物镜（NA 1.4），氩（488纳米）和氦氖（543纳米，633纳米）激光器和蓝色二极管（405纳米）。 </li><li>将鼠标与盖玻片覆盖肝脏面朝下放在客观的显微镜阶段。设置在腔室的温度升高到29℃。使用肝自发荧光，以允许血窦可视调整焦点。 </li><li>为了显现血管，制备500微克的BSA-Alexa的647中的溶液稀释在100μlPBS中，每只小鼠。静脉内注射该溶液到尾静脉麻醉小鼠用1ml注射器与地下30针。 </li><li>为了显现肝细胞核，制备的Hoechst 33342的在PBS中的溶液。注入此溶液在8毫克/公斤的小鼠腹膜内用1ml注射器与地下30针。 </li><li>使用正常的顺序方式，63X浸没物镜和扫描仪在400赫兹到图像肝细胞核和肝脉管。打开405纳米的蓝色二极管激发赫斯特和BSA-647 Alexa的激发633纳米的激光。打开氩488 nm激光上定义大量收购乐队以可视化的肝自体荧光。 </li></ol>肝脏血流速度测量的活体4显微成像<ol><li>打开显微镜（LAS-AF浏览器版本3.1.0版本8587）的LAS软件。打开显微镜软件时选择共振扫描仪模式。 </li><li>单击配置选项卡上设置硬件。点击激光，然后选择氦氖激光633。点击设置并选择12位分辨率。 </li><li>点击获取菜单上。在光束路径设置窗口中，选择目标63X，并成立了633激光功率为100％。激活信号增益和关闭设定的参数从PM1-3下拉菜单中选择Alexa的-633。 </li><li>在采集选项卡中，选择“XYT”收购模式。设置格式宽1024×1024。在8000赫兹选择速度。选择3艾里单位针孔。选择3，缩放因子不要选择双向模式。 </li></ol> RBC速度的使用XYT图片5.定量分析（方法1） <ol><li>点击“在线”图标，在可视化的实时模式的血流量。选择感兴趣的区域，并选择一个特定的血管进行分析。通过调整USB控制面板上的“X”，“Y”和扫描磁场旋转坐标设定感兴趣的水平位置的容器中。 </li><li>停止“现场”模式，一旦感兴趣的容器设置。更改格式设置宽度为1024×256的采集模式设置窗口，平均线= 1，帧平均= 1，选择“栈”150点击“开始”，以获取图像。 </li><li>对于使用红细胞速度的定量分析“XYT”图片打开ImageJ的软件。选择“文件”在ImageJ的选项卡，单击“打开”，然后选择感兴趣的文件。 </li><li>转至ImageJ的“插件”菜单，选择基因座和生物格式导入选项。在这个窗口中，选择查看栈参数“hyperstack”。在堆叠顺序选项，选择该选项：“xyzct”。注意：这将打开一个窗口，所有的一系列收购。 </li><li>点击ImageJ的菜单上的“插件”，然后选择MTrackJ选项。点击“添加”图标上的小窗口，点击RBC跟踪。点击相同的红细胞中的每个以下框架，然后单击“度量”图标来测量速度。 注：生成可以保存在.exl格式的文件。 </li><li>跟踪至少每容器5 RBC和分析至少相同大小的三个单独的容器中。 </li></ol> RBC速度的使用XT线路图像6.定量分析（方法2） <ol><li>点击“直播”进行可视化血˚F低在实时模式。选择一个特定的血管进行分析。设定感兴趣的水平位置的容器中。停止“现场”模式，一旦感兴趣的容器设置。 </li><li>选择“XT&#39;扫描模式，并设置沿行扫描所选容器的中心内腔中。定格宽1,024×512，速度= 8,000Hz，平均线= 32，时间= 512。点击“开始”，并获得XT线路图像。 </li><li>通过打开.lif文件，并打开感兴趣的XT线图像产生条纹的红细胞速度的定量分析。在LAS-AF软件，选择“实验”，打开.lif并选择图像。注意：它会自动打开一个kymograph。 </li><li>测量所需的颗粒条纹（表示暗反射）的时间（t，获得的在Y轴）的旅行有关此kymograph一定距离（d，所得的X轴）。选择工具“画线”水平画一条线条纹。注意在远处微米，在图像的底部中的结果标签产生秒的时间。 </li><li>计算速度为使用从kymograph获得的值：V =距离/时间。 </li><li>量化最少每XT图像5颗粒条纹和最小的尺寸相同的三个单独的容器中。 </li></ol>

<ol>
	<li>Vollmar, B., Menger, 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=The+hepatic+microcirculation:+mechanistic+contributions+and+therapeutic+targets+in+liver+injury+and+repair.">The hepatic microcirculation: mechanistic contributions and therapeutic targets in liver injury and repair.</a> Physiol. Rev. 89, 1269-1339 (2009).</li><li>Srinivasan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+blood+flow+measured+by+optical+methods.">Absolute blood flow measured by optical methods.</a> SPIE Newsroom. , (2011).</li><li>Seifalian, A. M., Stansby, G. P., Hobbs, K. E., Hawkes, D. J., Colchester, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+liver+blood+flow:+a+review.">Measurement of liver blood flow: a review.</a> HPB. Surg. 4, 171-186 (1991).</li><li>Engwerda, C. R., Ato, M., Kaye, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages,+pathology+and+parasite+persistence+in+experimental+visceral+leishmaniasis.">Macrophages, pathology and parasite persistence in experimental visceral leishmaniasis.</a> Trends Parasitol. 20, 524-530 (2004).</li><li>Beattie, 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=Dynamic+imaging+of+experimental+Leishmania+donovani-induced+hepatic+granulomas+detects+Kupffer+cell-restricted+antigen+presentation+to+antigen-specific+CD8+T+cells.">Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.</a> PLoS Pathog. 6, e1000805 (2010).</li><li>Lee, W. Y., 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=An+intravascular+immune+response+to+Borrelia+burgdorferi+involves+Kupffer+cells+and+iNKT+cells.">An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells.</a> Nat. Immunol. 11, 295-302 (2010).</li><li>Geissmann, 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=Intravascular+immune+surveillance+by+CXCR6++NKT+cells+patrolling+liver+sinusoids.">Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids.</a> PLoS Biol. 3, e113 (2005).</li><li>Marques, P. 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+liver+biology+in+vivo+using+conventional+confocal+microscopy.">Imaging liver biology in vivo using conventional confocal microscopy.</a> Nat. protocols. 10, 258-268 (2015).</li><li>Thiberge, 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+imaging+of+malaria+parasites+in+the+murine+liver.">In vivo imaging of malaria parasites in the murine liver.</a> Nat. protocols. 2, 1811-1818 (2007).</li><li>Vacchina, P., Morales, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+screening+test+using+Leishmania+promastigotes+stably+expressing+mCherry+protein.">In vitro screening test using Leishmania promastigotes stably expressing mCherry protein.</a> Antimicrob. Agents Chemother. 58, 1825-1828 (2014).</li><li>Ferrer, M., Martin-Jaular, L., Calvo, M., del Portillo, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+spleen:+quantitative+analysis+of+parasite+mobility+and+blood.">Intravital microscopy of the spleen: quantitative analysis of parasite mobility and blood.</a> J. Vis. Exp. , (2012).</li><li>Kamoun, W. 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=Simultaneous+measurement+of+RBC+velocity,+flux,+hematocrit+and+shear+rate+in+vascular+networks.">Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks.</a> Nat. Methods. 7, 655-660 (2010).</li><li>MacPhee, P. J., Schmidt, E. E., Groom, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittence+of+blood+flow+in+liver+sinusoids,+studied+by+high-resolution+in+vivo+microscopy.">Intermittence of blood flow in liver sinusoids, studied by high-resolution in vivo microscopy.</a> Am. J. Phys. 269, G692-G698 (1995).</li></ol>

The authors have nothing to disclose.

小鼠肝脏的活体显微镜的最近发展开辟了在体内和实时5,9,10生理响应于感染的调查新的可能性。器官的血流量是一个关键的生理参数是经常改变的许多疾病。然而，生理和传染性条件下肝脏血流动力学的状态仍然是一个探索不良的区域。在这项研究中，IVM为基础的方法，这在以前适于脾和肿瘤脉管系统的研究中，实施了以量化在单个容器内肝脏血流速度，并测量在体内 11...

器官特异性血流动力学是任何哺乳动物器官重要的生理功能。血流异常可能是炎症的后果和器官功能障碍1的标志。因此，血液流动的组织，结构和功能出现生理和病理条件下进行分析的关键参数。已普遍用于分析在特定器官的血流量的技术包含一些限制，包括技术本身的分辨率极限（ 例 ​​如血流的多普勒成像），为的绝对血液流量只（体积的血液每次测量的能力单元服务器官）（ 例如光学相干断层扫描）和在速度在大量异质群体血管2,3的平均变化的测量。肝脏的循环系统链接不同容器亚型是异质在它们的大小，结构和功能。在这项研究中，活体显微镜（IVM）成像技术被应用到评估肝脏血流动力学体内，实时，在高分辨率和在平行于揭露的组成该肝器官的个体的血管的特征。这个强大的光学成像技术的最新发展使研究人员收集的动物生活在高时空分辨率的动态数据。通过允许直接可视化和体内特异性和快速生物过程的实时监控，IVM提供了一个独特的机会，研究者将图像的个别血管，并测量和量化单个红血球内的特别选定的速度（RBC）肝血管。 在这项研究中，我们已经实现了在小鼠肝脏的IVM技术研究小鼠感染的肝血流动力学的嗜肝寄生虫利什曼原虫的影响。L.原虫负责内脏利什曼病，严重的疾病，其特征急性上慢性炎症反应和病理学，其存在于多个器官，包括脾和肝脏中的试剂。内脏利什曼病的实验小鼠模型中，肝感染是自我解决，而脾感染是渐进4。 利什曼原虫属感染的相对于该个体的器官，这些结果仍然没有完全了解。肝，脾血流动力学病理状态下调查将揭示宿主相互作用的寄生虫和疾病发病机制的新光源。 我们的实验模型系统是基于曝光和成像接受静脉注射特异性的荧光染料对肝intravasculature的标记麻醉小鼠的肝脏。肝脏是一个有利的器官内活体显微镜。执行一个小incisio后中的n腹部，肝脏轻轻外在并置于湿纱布，然后在与减少由于心跳和呼吸的任何运动伪影的目标盖玻片。肝脏，然后放置在显微镜透镜的视图内。相比于需要使用两个光子显微镜对IVM研究脾和淋巴结，肝的优势在于其均质3D架构/解剖学，允许使用常规共焦显微镜，具有最大穿透深度约50微米，对于活体显微镜成像5-8。 这项研究描述了两个独立的成像方法是在个别血管红细胞流速和血流速度的定量测量。在第一种方法中，使用的是XY二维的模式随时间获取肝脏血流量。所得XYT数据使用MtrackJ插件在自由的ImageJ软件，其允许individ的跟踪分析UAL红细胞随着时间的推移。在第二种方法中，一个单一的血管被选择及其相应的血流是利用共焦激光扫描显微镜的线扫描快速采集模式进行分析。感兴趣的容器被扫描在沿其中心轴的高频通过的轴线。血流速度，然后根据在未标记暗红细胞和荧光标记的等离子体之间的对比度的差量化。沿着线扫描中获得的红细胞和血浆的荧光强度对时间作图，得到的条纹，角度，其中成比例的单个红细胞的速度。 本文的目的是提供肝个别血管内的简单和可再现的方法进行成像并测量血流速度，并备妥的基本工具小鼠手术，IVM和速度的定量分析的成功表现个别红细胞。 Ť他的做法将允许研究人员获得病理状态下新的见解血流速度。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-Alexa 647</td>
			<td>lifetechnologies</td>
			<td>A34785</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran-FITC 500 mol wt</td>
			<td>SIGMA</td>
			<td>46947</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>PanPharma</td>
			<td>20434</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>KP07KEU</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetedine</td>
			<td>Pharma Animal</td>
			<td>6869029</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate liquid</td>
			<td>Cyanolit</td>
			<td>5833300005</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip frame: Membrane slide for microdissection part N&#176;: 5013</td>
			<td>Molecular machines</td>
			<td>50103</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip DiaPath 24x60 ep: 1.6 mm</td>
			<td>DiaPath</td>
			<td>61061</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica&#160;</td>
			<td>TCS-SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS-AF viewer&#160;</td>
			<td>Leica software</td>
			<td>Version 3.1.0 buid 8587</td>
			<td></td>
		</tr>
	</tbody>
</table>

intravital microscopy imaging of the liver following leishmania infection: an assessment of hepatic hemodynamics

活体显微镜（IVM）是已经成为可能在实时和在活体动物各种生物事件的可视化，监测和量化一个强大的光学成像技术。该技术极大地提高了我们的生理过程，并在特定器官病原体介导的现象的理解。 在这项研究中，IVM施加到小鼠肝脏和协议设计为图像在体内肝脏的循环系统，并测量在个体肝脏血管的红血细胞（RBC）的速度。为了显现不同容器亚型表征肝器官和执行血流速度的测量，之C57B1 / 6小鼠进行静脉内注射一种荧光等离子体试剂标签的肝相关脉管。 IVM能够在体内实时，红细胞速度的测量中感兴趣的特定容器中。建立这一方法将使得能够研究生理和病理条件下肝血流动力学。最终，这种基于成像的方法将是非常重要的研究L的影响原虫感染对肝脏血流动力学。 这种方法可以适用于其它感染模型和小鼠的器官和可能进一步扩展通过量化其对血流量的影响预临床的炎症的药物的效果的测试。

在肝血窦的特定的架构组织可以基于这个器官（ 图1，小图B和C，绿色），腹膜内注射的Hoechst的肝细胞核的标记的自身荧光属性可视（ 图1B，蓝）和静脉注射荧光牛血清白蛋白为肝循环系统（ 图1C，红色）的染色。肝脏是由若干不同容器亚型具有不同的功能和结构以及尺寸范围从40-700微米2直径（D）的（小血管D是间40-80微米2和大血管D是大于300...

Watch this Scientific Journal Video about Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics at JoVE.com

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

肝脏活体如下显微成像<em&gt;利什曼原虫</em&gt;感染：肝脏血流动力学的评估

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

intravital-microscopy-imaging-liver-following-leishmania-infection-an

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

11.4K 次观看.  University of Aix-Marseille. This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

视频: Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

腹股沟淋巴结的活体显微镜

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

小鼠腘窝淋巴结的活体成像

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...