The authors thank the Central Animal facility of the University Hospital Aachen for technical support. This work was supported by the German Research Foundation (DFG Ta434/2-1, DFG SFB/TRR 57) and by the Interdisciplinary Center for Clinical Research (IZKF) Aachen. This work was further supported by the Core Facility &#8221;Two-Photon Imaging&#8221;, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University.

注：该实验按照管理下的“指南实验动物的护理和使用”（NIH出版，第8版，2011）动物研究的德国法律执行和动物保护的指令六十三分之二千零一十/ EU用于科学目的（官方公报欧洲联盟，2010年）。官方许可从政府的动物护理和使用办公（LANUV北威州，雷克林豪森，德国），视为理所当然。 注意：步骤可以为短期成像省略（ 例如卡扣镜头，三维堆叠或还短的持续时间的时间推移显微镜）的标有星号（*），以减少准备时间和简化手术协议。也可以不广泛的监测和控制循环执行成像如果有必要，但是，生存时间会明显减少。 1.显微镜设置和手术前的准备（5-10英里N） <ol><li>开关系统（显微镜，电源实验室，激光，加热垫，气候室，网络摄像头，注射泵设备，呼吸器）。 </li><li>连接的O 2供给到异氟烷蒸气，并设置电平异氟烷1.5％（体积）（体积/体积）中，连接到一个小动物呼吸器。 <ol><li>短期成像，只在最初的IP麻醉诱导使用异氟醚维持麻醉（见步骤2.2）。然后用2体积％（体积/体积）异氟醚，并且保持成像时间低于2小时，以保证稳定的肝脏微循环。 </li></ol></li><li>设置呼吸到120-140呼吸周期/分钟150-200微升的体积。 </li><li>设置鼠标琼脂糖阶段。制备100ml的3％（重量/体积）琼脂糖，倒入在40°的角度的盘（约10cm×15cm的基体）。 </li><li>成立了手术工作区域收集所需要的仪器仪表。防止微生物感染，消毒使用Sekusept复S的1％溶液（文书9.9％w / v的），戊二醛9.8％w / v的，甲醛的实验（ 图1A前5分钟）。 </li><li>获得剩余组分：皮肤消毒剂（ 例如 ，10％聚维酮碘溶液），肝期（栈＆桥），琼脂糖溶液（3％（重量/体积）的PBS），注射泵，用5％（重量/体积）的葡萄糖溶液（G-5），注射泵用麻醉剂溶液I（氯胺酮0.1毫克/毫升，甲苯噻嗪0.5毫克/毫升，芬太尼5微克/毫升），棉签，正丁基-2- Cyanoacrylat，和1x PBS中。把琼脂糖阶段菜进入孵化室预热。 </li></ol> 2.气管切开术（10-15分钟） <ol><li>从在房子育种称量25-28克使用C57BL / 6小鼠。房子无特定病原体条件下按照FELASA指南，在一个温度和湿度可控的环境中以12小时光照/ 12小时黑暗周期下的小鼠。让动物自由出入标准小鼠饲料（嗅嗅标准啮齿类动物食物），自由采食和饮水。 </li><li>麻醉鼠标由init胶质腹膜内（IP）注射250微升麻醉剂溶液II（氯胺酮0.1毫克/毫升，甲苯噻嗪1毫克/毫升，丁丙诺啡10微克/毫升）。 <ol><li> *对于在活体成像的维持，连续地通过连续腹腔注射施用麻醉剂溶液I（氯胺酮0.1毫克/毫升，甲苯噻嗪0.5毫克/毫升，芬太尼5微克/毫升）使用注射泵装置为0.2毫升/小时的流速用吸入性异氟烷0.5％（体积）的组合（体积/体积）。 </li><li>检查反射5分钟（ 如捏垫脚用钳子）后，皮肤消毒气管切开，开始准备，如果鼠标在手术麻醉的耐受状态。 </li><li>适用于眼部防护霜（ 如泛醇50毫克/克），以防止角膜变干（ 图1B）。 </li></ol></li><li>注视鼠标放在准备表暴露腹侧使用的四肢胶带;轻轻过度伸张颈部，固定在这个位置上， 例如 ，通过使用橡胶带钩住吨澳门牙。 <ol><li>使用聚维酮碘溶液，通过施加消毒剂用棉签消毒皮肤和毛皮。 </li></ol></li><li>执行最初的皮肤切口（0.5〜1厘米长）的正下方的下巴（ 图1C） <ol><li>小心解剖唾液腺（ 图1D）之间的结缔组织。 </li><li>小心撕开肌性管道周围的气管（ 图1E，F）。 </li></ol></li><li>将手术线气管的后通风管固定（ 图1G）下方。 <ol><li>与进行T形切口软骨环之间微型剪刀打开气管（ 图1H） </li></ol></li><li>将通风管进入气管通过切口（约0.5厘米）。要管推进，抢尾端的小镊子解剖，轻轻推进管插入气管（ 图1I） </li><li>注视管手术线（ 如 </eM&gt;，5-0）螺纹放置在步骤2.5绑管周围结气管内;在皮肤进行管的第二颅固定，以避免意外的撤离（ 图1J，K）。 </li><li>密封切割用组织胶（ 例如 ，正-丁基-2- Cyanoacrylat）（ 图1L）。 </li><li>固定管在头用胶带。 </li></ol> 3.开腹（15-20分钟） <ol><li>放在加热垫的鼠标，以防止体温过低。剃腹部，小心地从皮肤去除毛发。通过使用聚维酮碘溶液消毒皮肤剃光和毛皮。 </li><li>进行小皮用切割手术剪（ 图2A）胸骨下方。延长横向切下面的肋骨两侧，烧灼所有可见的血管，防止出血。 </li><li>认真履行一个小切口，在胸骨下方的白线，打开腹膜（ 图2B）。延长削减双方使用cauterizATION防止出血（ 图2C，D）。 </li><li>将鼠标放到琼脂糖阶段菜（ 图2E）。放置成堆的适当高度上的鼠标（通常12-14盖玻片）（ 图2F）的两侧上。 </li><li>广场上背部下方的呼吸触发传感器同步的呼吸显微镜。激活触发单元（ 图2G）。 </li><li>通过胸骨将手术线（5-0）收回肋骨（ 图2I）。 </li><li>小心使用弯曲的手术剪刀剪韧带连接肝脏和隔膜（镰状韧带），以及肝和胃肠道。切镰状韧带下到主动脉（ 图2J）。 </li></ol> 4.样品设置（10-15分钟） <ol><li>将鼠标放在右侧以45°为方便大肝叶的角度。 </li><li>添加分期桥下面的肋骨，覆盖腹部腔。通过使用标准的盖玻片用胶带涂层以覆盖尖锐边缘（ 图2K）制造的桥梁。 </li><li>将大肝叶在舞台上。小心滑双球触笔探针或肝脏低于衬垫刮刀和保持利用湿式棉签或湿纸巾的器官的顶部。提起叶到幻灯片和应用温和的阻力。叶可弯曲或折叠。提供额外的小心，只温柔的操控肝脏（ 图2L）中。 </li><li>放置侧向支承树桩， 例如 ，小的盖玻片（20毫米×20毫米），它旁边的肝叶的大致相同的高度桩支撑盖玻片。 </li><li>将大盖玻片（24毫米×50毫米）的肝叶。确保盖玻片是面向尽可能的水平（ 图2M）。 注：盖玻片应该在与组织接触而不挤压它。检查受损的血流量（白色组织）的明显迹象。如果microcirculation被打乱，进一步增加支持的股份。 </li><li> *将两个独立的腹腔导管（ 图2N）长期麻醉和G-5的应用程序。横向下腹部安装导管旁边的后肢。 注：腹膜内导管可自由连接27G的针，柔性硅树脂管（ 图3）。 </li><li> *固定针用5-0手术线在皮肤上的循环，以避免意外的位移。 </li><li> *附加​​注射泵麻醉解决方案，我到导管1，设置流速0.2毫升/小时;附加注射器泵与G-5到导管2，设置流速0.1毫升/小时。 </li></ol> 5.鼠标监测<ol><li> *将ECG电极成前部和后端部（ 图4A）。 </li><li> *附加到期油管CO 2液位传感器。 </li><li>添加外部温度传感器连接到散热垫（ 图4C）。 </li></ol> 6.嵌入和组织固定（5-10分钟） <ol><li>制备100ml的3％（重量/体积）琼脂糖在1X PBS中。嵌入肝时温度为41°C使用5毫升注射器和18G的针头（ 图5A）。 </li><li>倒入其余琼脂糖上盖玻片和周围的小鼠（ 图5B，C）。等到琼脂糖完全糊化。 </li><li>删除使用海德曼抹刀过量琼脂糖，制备足够一个视场大的扫描准备肝叶（ 图5D中的E）。 </li></ol> 7.成像<ol><li>转移鼠标放到显微镜气候室。 </li><li>加50-100毫升的1×PBS的（预热37℃）。 </li><li>覆盖样品盘，以防止蒸发（ 图5F）。 </li><li> *缩小吸入性异氟烷〜0.5体积％（体积/体积）。 </li><li> *启动监控软件，记录心电图，心脏率和呼气CO 2。 </li><li>启动成像：开拉SER百叶窗，确定视场的利益，定义为Z-栈上下边界，并且开始时间推移录音。调整的Z堆叠如果需要校正的Z-漂移（ 例如 ，由于血压变化或温度波动， 如图（5G）。 注意：在摄像期间的动物的循环或麻醉的，或者如果完成变得不稳定，通过颈脱位（没有觉醒从麻醉）牺牲小鼠。 </li></ol>

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The authors disclose no con&#64258;ict of interests.

我们研究的目的是开发一个高度标准化的，稳定的和可重复的方法肝脏活体成像TPLSM。一般活体成像给以下归巢和发展，动态平衡和疾病的不同白细胞群体的互动有价值的见解在现实生活条件的细胞行为。但是，肝脏的几分有挑战性解剖位置，由于其呼吸道和肠道蠕动运动直接被传递到肝以及它们的高脆性相对于其他固体器官施加用于稳定长期活体成像几个挑战。近年来，许多实验研究小鼠揭示?...

细胞和在整个器官的细胞功能或甚至整个生物体的可视化受到了极大的兴趣超过50年，包括本体2的几乎所有的部分。因此，一些早期的研究已经采用的肝3,4的活体成像。但是，有几个局限存在最新关于长期稳定的高分辨率的肝组织的成像。 由于在与膜片和胃肠道5紧密接触肝脏的解剖位置，因为微观活体成像的最常见的问题是移动由于呼吸和，在较小程度上，蠕动肠道6。相较于其他实体器官，肝脏手术是特别具有挑战性。由于密集的微血管结构，手术操作可导致大量出血病灶，居民受损的微循环7，也激活我mmune细胞如Kupffer细胞8。因此，该组织的机械固定如别处公布6,9-可能妨碍与活体显微镜成像。 在一个健康的肝脏，总血量的10-15％驻留在肝脉管系统中，并且该机关收到整体心输出10的25％左右，使该器官极易受到在循环变化（ 例如 ，血压波动）。因此，由于例如 ，剪切应力，位移，伤害由过度的组织处理或集中式循环中断的肝血流量将导致人工改变在白细胞迁移行为，受损的肝的氧合，因此进一步的肝损伤，影响肝的免疫反应，以及作为器官保存和动物的整体使用寿命。 早期的微观研究是基于活体萤光英里croscopy，但一些技术限制，如光漂白和低渗透深度限制使用这种技术的长期的肝脏成像4,11,12。在20世纪90年代多光子显微术的发展，光漂白或穿透深度的限制，主要是解决了，因为这种新的方法，在技术上是能够在根据实际生活情况13-15几乎所有的器官进行成像研究。然而，对于肝脏成像主剩余的挑战是：呼吸运动，肝组织的自体荧光，确保为几个小时，16长时间不变的血流量在肝窦，特别是稳定的成像。 尽管一些研究涉及的功能和各种白细胞的迁移在肝脏17， 如 NKT细胞18-20，T细胞21,22，肝巨噬细胞23,24或25的中性粒细胞，长期的多光子米icroscopy成像尚未成功建立，任务更有挑战性的动物的急性或慢性肝病，由于现有的损害，因此，较高的敏感性，以进一步的损害26。然而，监测在实时肝白细胞的迁移行为和细胞功能允许在其特定的作用，在肝脏内稳态和疾病27新的见解。 趋化因子受体CXCR6上表达几种淋巴细胞亚群，包括自然杀伤（NK）细胞，NKT细胞和一些T细胞群18,28。在小鼠中之前的研究已经表明，CXCR6和其同源配体CXCL16可能动态平衡期间控制NKT细胞的巡逻肝血窦。因此，使用CXCR6.gfp小鼠（携带一敲，在绿色荧光蛋白[GFP]在CXCR6基因）被描述为调查淋巴细胞迁移的各种器官如脑29并且还肝脏18,20，示出了在炎症增加CXCR6.gfp细胞浸润。 在这项研究中所提供的方法有可能在一段时间稳定的条件下长时间遵循这些进程。活体多光子基于程序允许成像，这是与动物的器官极小扰动高度重复性;长期生存的动物被大量监控后严密控制呼吸和循环的优化;高度灵活，也容易采取其他实质器官如肾脏或脾脏。

<table border="0" cellpadding="0" cellspacing="0" width="1184">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Anesthetics</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td style="width:293px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Buprenorphine</td>
			<td style="width:263px;">Essex Pharma</td>
			<td style="width:219px;">997.00.00</td>
			<td>Analgeticum, 0.1 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fentanyl</td>
			<td style="width:263px;">Rotex Medica</td>
			<td style="width:219px;">charge: 30819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fluovac anesthesia system</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td style="width:219px;">34-1030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Glucose 5%</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ISOFLO (Isoflurane Vapor) vaporiser</td>
			<td style="width:263px;">Eickemeyer</td>
			<td style="width:219px;">4802885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Isoflurane</td>
			<td style="width:263px;">Forene Abbott</td>
			<td style="width:219px;">B 506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Isotonic (0.9%) NaCl solution</td>
			<td style="width:263px;">DeltaSelect GmbH</td>
			<td style="width:219px;">PZN 00765145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Ketamin 10%</td>
			<td style="width:263px;">ceva</td>
			<td style="width:219px;">Charge: 36217/09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Xylazin 2%</td>
			<td style="width:263px;">medistar</td>
			<td style="width:219px;">Charge: 04-03-9338/23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Consumable supplies</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">20ml Syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250ml Erlenmeyer flask</td>
			<td style="width:263px;">Schott Duran</td>
			<td>21 226 36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">25mL Beaker 2x</td>
			<td style="width:263px;">Schott Duran</td>
			<td style="width:219px;">50-1150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">2ml syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl suture</td>
			<td style="width:263px;">Ethicon</td>
			<td style="width:219px;">V7980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Agarose</td>
			<td style="width:263px;">commercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Bepanthen Eye and Nose ointment</td>
			<td style="width:263px;">Bayer Vital GmbH</td>
			<td style="width:219px;">6029009.00.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Change-A-Tip Deluxe High-Temp Cautery Kit</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">18010-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cotton Gauze swabs</td>
			<td style="width:263px;">Fuhrmann GmbH</td>
			<td style="width:219px;">32014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cover Slip 24x50mm</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1871</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Durapore silk tape</td>
			<td style="width:263px;">3M</td>
			<td style="width:219px;">1538-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Feather disposable scalpel</td>
			<td style="width:263px;">Feather</td>
			<td style="width:219px;">02.001.30.011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fine Bore Polythene Tubing 0,58mm ID</td>
			<td style="width:263px;">Smiths medical</td>
			<td style="width:219px;">800/100/200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Histoacryl</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;">1050052</td>
			<td>5x 0,5ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Leukoplast</td>
			<td style="width:263px;">BSN Medical Inc.</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Microscope Slides</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Poly-Alcohol Haut&#8230;farblos Antisepticum</td>
			<td style="width:263px;">Antiseptica GmbH</td>
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			<td height="21" style="height:21px;width:411px;">Sterican needle 18 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">304622</td>
			<td></td>
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			<td height="21" style="height:21px;width:411px;">Sterican needle 27 3/4 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">4657705</td>
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			<td height="21" style="height:21px;">Dumont No.7 forceps</td>
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			<td height="21" style="height:21px;width:411px;">Small surgical scissors curved</td>
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			<td style="width:219px;">14029-10</td>
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			<td height="21" style="height:21px;width:411px;">Small surgical scissors straight</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14028-10</td>
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			<td height="21" style="height:21px;width:411px;">Standard pattern forceps</td>
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			<td height="21" style="height:21px;width:411px;">ECG Trigger Unit</td>
			<td style="width:263px;">Rapid Biomedical</td>
			<td style="width:219px;">3000003686</td>
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			<td height="21" style="height:21px;width:411px;">MICROCAPSTAR End-Tidal Carbon Dioxide Analyzer</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Minivent Typ 845</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td>73-0043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Multiphoton microscope Trimscope I</td>
			<td style="width:263px;">LaVision</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Perfusor Compact</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">PowerLab 8/30 8 channel recorder</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;">PL3508</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature controlled heating pad</td>
			<td style="width:263px;">Sygonix</td>
			<td style="width:219px;">26857617</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature sensor</td>
			<td style="width:263px;">comercially available</td>
			<td style="width:219px;"></td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature controlled System for Microscopes -Cube&#38;Box</td>
			<td style="width:263px;">Life Imaging Services</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using cxcr6.gfp reporter mice

<html>肝脏炎症作为对损伤的响应是，涉及白细胞的不同的亚型，包括单核细胞，嗜中性粒细胞，T细胞亚群，B细胞，自然杀伤（NK）和NKT细胞的浸润高度动态的过程。肝脏用于监测免疫细胞迁移的活体显微镜是特别具有挑战性的，由于有关样品制备和固定，光学分辨率和动物长期存活的高要求。然而，炎症过程以及细胞相互作用研究的动力学能提供更好的理解炎性肝病的发生，发展和回归的关键信息。因此，建立了一个高度敏感的和可靠的方法来研究的迁移和不同的免疫细胞在小鼠肝脏在长时间内（约6小时），通过活体双光子激光扫描显微镜（TPLSM）细胞 - 细胞相互作用中有重症监护组合监控。 ENT“&gt;所提供的方法包括：一个轻柔的制备和与器官的极小扰动肝脏的稳定固定;使用多色多光子显微镜，几乎没有光漂白或光毒性效果在长达6小时的时间段长期活体成像，允许跟踪具体的白细胞子集;而由于大量监测小鼠重要参数和流通，温度和气体交换的稳定稳定的成像条件。 调查在肝脏炎症淋巴细胞迁移CXCR6.gfp敲入小鼠下基线条件和后诱导四氯化碳腹腔注射（多个）（CCL 4）的急性和慢性肝损伤进行活体肝成像。 CXCR6是表达在淋巴细胞趋化因子受体，主要对天然杀伤T（NKT） - ，天然杀伤（NK） - 和T淋巴细胞，如CD4 + T细胞亚群，而且粘膜associated不变（MAIT）T细胞1。继允许在肝损伤的详细洞察他们的行为改变，因此他们的病情恶化可能参与的迁徙模式CXCR6.gfp +免疫细胞和定位。

为了验证我们的活TPLSM方法，我们受到CXCR6 GFP / +小鼠活体成像TPLSM。小鼠要么不及时治疗作为基线控制或受到单一腹腔注射四氯化碳（CCL 4）诱发急性肝功能损害20。 视频序列进行了接管2-5小时的时间段，并将细胞追踪随着时间的推移，由于其绿色荧光。来显示普通细胞运动，在操作过程中检测到的所有轨道被绘制为叠加后的图像（ 图6A）。</stro...

Watch this Scientific Journal Video about Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice at JoVE.com

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

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, ...

long-term-intravital-multiphoton-microscopy-imaging-immune-cells

Research

JoVE Journal

Immunology and Infection

11.8K 次观看.  RWTH University-Hospital Aachen. 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.

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

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

科研

免疫与感染

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 ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

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 ...

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

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

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

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

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

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

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

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

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 ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

使用IL-1β启动子驱动的红色荧光蛋白记者小鼠中性粒细胞引发的活体成像

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...