Name: long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using cxcr6.gfp reporter mice
Uploaded: 2015-03-24T15:00:00.000+00:00
Duration: 11 min 44 s
Description: 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

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了免疫细胞浸润高度动态性质伤肝28，与居民或浸润单核/巨噬细胞30-32，中性粒细胞33或各种淋巴细胞亚群34,35作出了重大贡献。然而，米这些数据的OST来源于终点分析（ 例如 ，多色牺牲动物后，流式细胞仪）。截至目前只有少数研究存在使用多色活体成像，描述在肝脏炎症细胞的流量的动态。使用倒置显微镜绕过必要性固定和肝脏的保湿由于组织到玻璃底24的牢固的粘合。然而，由于许多多光子显微镜构建为直立成像系统，还需要更复杂的制备和固定方法，以稳定的组织进行成像的时间。通常，定制分期系统已经描述用于组织固定6，36,37，但是与这些关键的是要验证肝脏微循环中，由于在组织上，即使最小的压力影响正弦血流甚至在患病肝脏38更严重的影响。 因此，以下几点是广告穿着该方法的设置，以优化得到的通过体内 TPLSM成像结果:增强制剂通过最小化样本扰动的微循环和中断的质量由于处理问题并增加动物存活;重症监护监测和麻醉，呼吸和循环进一步增强动物的生存稳定和如最小的生理假象，随后的适应，造成血液中的PH值不平衡。在带有触发成像组合控制呼吸是基本消除由于与呼吸周期的显微镜的同步移动工件。在含有生理盐浓度琼脂糖器官的嵌入有利于轻轻固定制备后的器官没有任何进一步的机械操作，也可防止样品脱水。用于肝显微镜最协议提供的方法只能站不足<html> BLE，长期麻醉。然而，在动物的存活可显著与设在这里，结合监测麻醉协议增强了足以保证存活多达8小时。可供选择的方法一般都适用于可视化的长达3小时的时间段内的过程，如嗜中性粒细胞浸润39，但缺乏的可能性，以图像更持久的过程，如单核细胞或淋巴细胞浸润22或甚至细胞增殖6。理解招募，渗透和细胞相互作用的动力学在急性和慢性肝脏炎症可提供更详细的洞察肝脏疾病的发生和发展。通过使用免疫病理学的这种增强的理解，将有可能设计出治疗更加精细在使用例如非选择性免疫抑制的方法处理所关注的靶细胞，而不是表示。 ove_content"&gt;要解决的白细胞的定位，轮回和相互作用，需要用于在细胞内的肝血管和肝脏组织18,20,40准确4D跟踪高质量的图像。 我们在这里提供的方法可以使用​​普通实验室试剂和设备得到​​应用，使其既易于处理和成本效益与样品的极小扰动。维持鼠标为尽可能长的生理条件下，有必要微调节呼吸体积和频率，以控制氧和血液的CO 2水平。虽然由于在连续输注的液体进入腹膜存在至少一个暂定供应稳定的循环，心电图和心脏频率测量只允许对循环稳定的局部控制。通过实现直接控制血压和中心静脉压液体应用很可能是至关重要的PARAMET器可以更稳定，从而导致进一步提高稳定性和存活的影响。 即使在高度控制的条件下，将动物的成像已经经受肝损伤模型中常用的小鼠，如对乙酰氨基酚引起的肝损伤， 四氯化碳诱发的肝损伤或饮食诱发的脂肪肝疾病仍然具有挑战性。由于肝脏中代谢状态的控制和其在循环中的中央位置的基本功能，肝功能或微循环的直接影响长期生存的动物，因此限制了可能的改变，以获得大量长TPLSM视频序列受损的器官， 如伴有严重出血或销毁微血管。另外，在极大地改变肝脏显微如结合广泛坏死模型，但它仍然难以研究细胞 - 细胞相互作用和炎症细胞聚集体的局部条块，因为没有斯坦了标准化"反染色"对于解剖结构（诸如苏木染色在传统的显微镜或DAPI染色在免疫荧光）可用于由成像方法进一步获得的数据的活TPLSM.The质量取决于细胞的标记强度和微观设置。 有几种方法存在以可视化的GFP +细胞在肝脏。 900和920 nm的激发波长之间获得最高的荧光具有非常低的背景荧光。肝背景荧光的几乎完全丧失不允许调查细胞的定位肝脏内，在不使用额外的容器跟踪诸如葡聚糖或凝集素。因此，我们决定图像的迁移白细胞在840nm的波长，给予GFP信号和详细，但不要太亮肝背景荧光之间的最佳比例。 综合来看，INTR在这项研究中引入AVITAL TPLSM成像系统可以应用于各种各样的肝特异性活体显微镜的问题。用这种方法，TPLSM成像在其他实体器官，如肾，肝，膀胱，肠或胰与的外科手术和组织分期只有轻微的修改是可行的，因此提供了高度灵活的方法中 ，调查细胞长期迁徙过程体内 。

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><tbody><tr><td>Anesthetics</td><td></td><td></td><td></td></tr><tr><td>Buprenorphine</td><td>Essex Pharma</td><td>997.00.00</td><td>Analgeticum, 0.1 mg/kg</td></tr><tr><td>Fentanyl</td><td>Rotex Medica</td><td>charge: 30819</td><td></td></tr><tr><td>Fluovac anesthesia system</td><td>Harvard Apparatus</td><td>34-1030</td><td></td></tr><tr><td>Glucose 5%</td><td>Braun</td><td></td><td></td></tr><tr><td>ISOFLO (Isoflurane Vapor) vaporiser</td><td>Eickemeyer</td><td>4802885</td><td></td></tr><tr><td>Isoflurane</td><td>Forene Abbott</td><td>B 506</td><td></td></tr><tr><td>Isotonic (0.9%) NaCl solution</td><td>DeltaSelect GmbH</td><td>PZN 00765145</td><td></td></tr><tr><td>Ketamin 10%</td><td>ceva</td><td>Charge: 36217/09</td><td></td></tr><tr><td>Xylazin 2%</td><td>medistar</td><td>Charge: 04-03-9338/23</td><td></td></tr><tr><td>Consumable supplies</td><td></td><td></td><td></td></tr><tr><td>20 ml Syringe</td><td>BD Plastipak</td><td></td><td></td></tr><tr><td>250 ml Erlenmeyer flask</td><td>Schott Duran</td><td>21 226 36</td><td></td></tr><tr><td>25 ml Beaker 2x</td><td>Schott Duran</td><td>50-1150</td><td></td></tr><tr><td>2 ml syringe</td><td>BD Plastipak</td><td></td><td></td></tr><tr><td>4-0 Vicryl suture</td><td>Ethicon</td><td>V7980</td><td></td></tr><tr><td>Agarose</td><td>commercially available</td><td></td><td></td></tr><tr><td>Bepanthen Eye and Nose ointment</td><td>Bayer Vital GmbH</td><td>6029009.00.00</td><td></td></tr><tr><td>Change-A-Tip Deluxe High-Temp Cautery Kit</td><td>Fine Science Tools Inc.</td><td>18010-00</td><td></td></tr><tr><td>Cotton Gauze swabs</td><td>Fuhrmann GmbH</td><td>32014</td><td></td></tr><tr><td>Cover Slip 24x50 mm</td><td>ROTH</td><td>1871</td><td></td></tr><tr><td>Durapore silk tape</td><td>3M</td><td>1538-1</td><td></td></tr><tr><td>Feather disposable scalpel</td><td>Feather</td><td>02.001.30.011</td><td></td></tr><tr><td>Fine Bore Polythene Tubing 0.58 mm ID</td><td>Smiths medical</td><td>800/100/200</td><td></td></tr><tr><td>Histoacryl</td><td>Braun</td><td>1050052</td><td>5x 0.5 ml</td></tr><tr><td>Leukoplast</td><td>BSN Medical Inc.</td><td></td><td></td></tr><tr><td>Microscope Slides</td><td>ROTH</td><td>1879</td><td></td></tr><tr><td>Poly-Alcohol Haut&amp;hellip;farblos Antisepticum</td><td>Antiseptica GmbH</td><td>72PAH200</td><td></td></tr><tr><td>Sterican needle 18 G x 1</td><td>B. Braun</td><td>304622</td><td></td></tr><tr><td>Sterican needle 27 3/4 G x 1</td><td>B. Braun</td><td>4657705</td><td></td></tr><tr><td>Tissue paper</td><td>commercially available</td><td></td><td></td></tr><tr><td>Surgical Instruments</td><td></td><td></td><td></td></tr><tr><td>Amalgam burnisher 3PL</td><td>Gatz</td><td>0110?</td><td></td></tr><tr><td>Blair retractors (4 pronged (blunt)) x2</td><td>Storz&amp;amp;Klein</td><td>S-01134</td><td></td></tr><tr><td>Dumont No.7 forceps</td><td>Fine Science Tools Inc.</td><td>91197-00</td><td></td></tr><tr><td>Graefe forceps curved x1</td><td>Fine Science Tools Inc.</td><td>11151-10</td><td></td></tr><tr><td>Graefe forceps straight x2</td><td>Fine Science Tools Inc.</td><td>11050-10</td><td></td></tr><tr><td>Heidemann spatula HD2</td><td>Stoma</td><td>2030.00</td><td></td></tr><tr><td>Needle holder Mathieu</td><td>Fine Science Tools Inc.</td><td>12010-14</td><td></td></tr><tr><td>Scissor</td><td>Fine Science Tools Inc.</td><td>14074-11</td><td></td></tr><tr><td>Semken forceps</td><td>Fine Science Tools Inc.</td><td>11008-13</td><td></td></tr><tr><td>Small surgical scissors curved</td><td>Fine Science Tools Inc.</td><td>14029-10</td><td></td></tr><tr><td>Small surgical scissors straight</td><td>Fine Science Tools Inc.</td><td>14028-10</td><td></td></tr><tr><td>Standard pattern forceps</td><td>Fine Science Tools Inc.</td><td>11000-12</td><td></td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools Inc.</td><td>15000-08</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>ECG Trigger Unit</td><td>Rapid Biomedical</td><td>3000003686</td><td></td></tr><tr><td>MICROCAPSTAR End-Tidal Carbon Dioxide Analyzer</td><td>AD Instruments</td><td></td><td></td></tr><tr><td>Minivent Typ 845</td><td>Harvard Apparatus</td><td>73-0043</td><td></td></tr><tr><td>Multiphoton microscope Trimscope I</td><td>LaVision</td><td></td><td></td></tr><tr><td>Perfusor Compact</td><td>B. Braun</td><td></td><td></td></tr><tr><td>PowerLab 8/30 8 channel recorder</td><td>AD Instruments</td><td>PL3508</td><td></td></tr><tr><td>Temperature controlled heating pad</td><td>Sygonix</td><td>26857617</td><td></td></tr><tr><td>Temperature sensor</td><td>comercially available</td><td></td><td></td></tr><tr><td>Temperature controlled System for Microscopes -Cube&amp;amp;Box</td><td>Life Imaging Services</td><td></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）。虽然基准条件下的细胞表现出长距离迁徙沿肝血窦向中央静脉，经过18小时四氯化碳治疗CXCR6 + / GFP的细胞显示出已经部分受损的运动模式。尽管一些快速移动的细胞仍然存在于肝血窦，几个重点领域是可见的与细胞从随机迁移到本地扫描已经改变，表明趋化反应招募细胞损伤的面积。效果更为明显36小时后，呈现出CXCR6 +细胞的几乎完全停滞，几乎没有活性迁移。而在基线条件下最迁移细胞具有可变的迁移速度与随机定向的改变， 四氯化碳处理的动物表现出的细胞只慢慢爬行以及一些自由游动细胞巡逻血窦后18小时，但不经过36小时。使用彩色编码的迁移轨迹，速度细胞可以直接可视评估的CCl 4处理的效果。受损的迁移也是在归一化的轨道的图（ 图6B）可见，显示出18小时后的CCl 4长距离的量跟踪以及所述平均磁道长度显着降低，而36小时后无游动细胞是可检测的任何更多。在线W第i这些发现，蜂窝位移，它描述一个小区的自由巡逻脉管的能力，随时间而减少，这表明经过36小时的CCl 4处理在肝细胞损伤（ 图6C）的部位的细胞的俘获。 遵循更详细CXCR6 + / GFP细胞中的肝的迁移行为，代表单个细胞的迁移速度作图形象化运动的水平随着时间的推移。我们和其他先前描述CXCR6 + / GFP细胞几种不同的运动模式:主动随机抓取与粘着滚动模式，显示出细胞运动和时间的静止状态的阶段;针对细胞与招聘细胞扫描感兴趣的领域，但仍然能够主动爬行;总细胞阻滞伴有免疫细胞活化18,20。虽然老鼠没有治疗主要表现细胞是自由莫蒂勒速度可达15微米/秒，CXCR6 + / GFP的细胞与CCl 4处理小鼠出现显着降低细胞的迁移速度和部分被捕后已经18小时，全迁徙被捕后36小时（ 图7A）。在与单细胞分析线，序列之内的所有单元的统计数据显示后的CCl 4处理（ 图7B），这也类似于由平均磁道的速度（ 图7C）降低总迁移速度和跟踪最大的迁移速度（ 图7D），表明该方法是适合于描述在发育肝疾病中的细胞迁移和定位的差异。 <img alt="图1" src="/files/ftp_upload/52607/52607fig1.jpg" /> 图1:鼠标的气管切开术（A）用于制备手术设备。 1X标准淅沥第N钳子，1个Semken钳，1个7号杜蒙钳，镊子格雷夫（锯齿，2个直/ 1X弯曲），2个布莱尔拉钩（4管齐下（冲服）），双滚珠笔探头（ 如银汞合金磨光3PL），海德曼锅铲HD2，持针器（马修或哈尔西），1个小型手术剪（锐/钝，1xcurved，1个直），微型剪刀（ 例如 ，Vannas弹簧剪刀），1小动物考特，棉签，眼睛防护药膏，手术螺纹，正丁基-2- Cyanoacrylatglue。 （B）的眼睛用眼保护软膏保护。 （C）初始皮肤削减气管准备。 （D）解剖颌下唾液腺的结缔组织。 （E）博览会气管。 气管周围肌肉组织的（F）解剖。手术螺纹（G）放置气管的管固定下面。 （H）的Trache人用切开软骨上部环之间microscissors。 （I）的插入换气管的进气管。颅外科螺纹（J）的位置，以固定管到皮肤上。 （K）管的双重固定。 （I）密封用正丁基-2- Cyanoacrylat手术切口。 <a href="https://www.jove.com/files/ftp_upload/52607/52607fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52607/52607fig2.jpg" /> 图2:开腹和肝制剂（A）初始切开皮肤，血管被烧灼，以防止失血过多。 （B）打开胸骨下方的腹膜。 （C）扩展使用烧灼单位腹膜切口。 （D）密封上腹船只。 （ <stron<html> g&gt;的E）低于肋骨腹膜切口的进一步扩展。 （F）安置动物到琼脂糖阶段菜。支持堆叠（F）安置。 （G）安置呼吸触发传感器。 （H）的触发阈值设置为呼吸同步成像。 （I）胸骨固定收回使用手术线肋骨。 （J）断开与镰状韧带的隔膜和解剖胆囊。分期盖玻片（K）放置。 （L）肝叶在舞台上放置。盖玻片上的肝叶的（M）安置。 IP导管长期麻醉（N）安置。 <a href="https://www.jove.com/files/ftp_upload/52607/52607fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html>图3"SRC ="/文件/ ftp_upload / 52607 / 52607fig3.jpg"/&gt; 图3:腹腔导管 27G的针头灵活的硅胶管连接。 <img alt="图4" src="/files/ftp_upload/52607/52607fig4.jpg" /> 图4:鼠标的监控。 心电图电极（绿，红，蓝线）（A）放置。针心电图检测放置皮下，如图所示。心电图（红色）（B）的监视，呼气的CO 2（蓝色）和心脏速率（红色）表示长期进展（左）和实际测量（右）。 （C）温度控制动力装置的散热垫的控制。温度设定为36℃。 <a href="https://www.jove.com/files/ftp_upload/52607/52607fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ve_content"FO:保持together.within页="总是"&gt; <img alt="图5" src="/files/ftp_upload/52607/52607fig5.jpg" /> 图5:肝嵌入和成像。 （A）嵌入肝叶的。在PBS 3％琼脂糖在41°C使用2毫升注射器带针18G盖玻片下注入。 （B）肝叶完全嵌入在琼脂糖，以尽量减少运动伪影。腹膜（C）密封剩余的琼脂糖，以防止脱水。 （D）去除观点琼脂糖覆盖显微镜领域的全糊化后。 （E）准备视场中。的Z级别，并使用光学显微镜的样品血流评估F的调整。 （D）盖盘，以防止水分蒸发过快。盖被提起，在图片显示设置。 <a href="https://www.jove.com/files/ftp_upload/52607/52607fig5highres.jpg" target="_blank">请点击此处查看大图</a>版本这个数字。 <img alt="图6" src="/files/ftp_upload/52607/52607fig6.jpg" /> 图6: 四氯化碳注射后跟踪CXCR6 GFP / +细胞在慢性肝损伤的小鼠腹腔注射0.6毫升/公斤的CCl 4溶解在向日葵油18小时或成像前36小时。在实验的当天，将小鼠如上所述经受活TPLSM。 （A）静态的细胞追踪图像。从各个时间点的轨道叠加，显示细胞运动和位移。使用单一光束泰坦萨费尔激光在840 nm和一个TPLSM显微镜拍摄图像。区域进行扫描以3-5的Z堆叠与渗透深度为70微米的具有每扫描周期约30秒的采样率。轨道是用颜色表示的迁移速度（浅蓝色:滞销或无柄细胞;紫色:游动细胞）。比例尺:100μ;米。 （B）的归一化对于其起源表示轨道方向和轨迹长度（微米）路径XY-图。从它们的起源细胞（C）总排量。 <a href="https://www.jove.com/files/ftp_upload/52607/52607fig6highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/52607/52607fig7.jpg" /> 图7:CXCR6 GFP / +细胞随后活TPLSM肝组织在基线和CCl 4处理的小鼠中的细胞对它们的迁移速度巡逻肝脏进行分析的迁移的统计信息 。单细胞（A）代表速度，随后随着时间的推移。速度测定在每个时间点，并绘制为每个帧单独地。线代表单个细胞。 Y轴是根据最大迁移速度缩放。 （B）圣从所有小区A.帧特定速度atistical分析进行分组并在基线分析和CCl 4处理的小鼠。 （C）平均轨道速度为每磁道和数据从所有轨道计算进行分组进行分析。每个轨道的（D）的最大速度作图和分析。所有实验均在3只动物的组进行，结果在两个独立的实验中得到证实。 *** P &lt;0.001（双尾非配对t检验）。 <a href="https://www.jove.com/files/ftp_upload/52607/52607fig7highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.9K 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

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

一个慢性双光子薄颅骨窗口技术在体内 Neuroinflammation模型小鼠小胶质细胞成像

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

科研

神经科学

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

通过透明硅胶窗进行纵向活体成像

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

癌症研究

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 ...

免疫与感染

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

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

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

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

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

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

医学

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 ...

Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue

Observing the intravascular dynamics of mouse liver tissue allows us to conduct further in-depth observations and studies on tissue-related diseases of the mouse liver. A mouse is injected with a dye that can stain blood vessels. To observe the mouse liver in vivo, it is exposed and fixed in a frame. Two and three-dimensional images of the blood vessels in the liver tissue are obtained using a multiphoton microscope. Images of the tissues at the selected sites are continuously acquired to observe long-term changes; the dynamic changes of blood vessels in the liver tissues are also observed. Multiphoton microscopy is a method for observing cell and cell function in deep tissue sections or organs. Multiphoton microscopy has sensitivity to tissue microstructure and enables imaging of biological tissues at high spatial resolution in vivo, providing the ability to capture the biochemical information of the organization. Multiphoton microscopy is used to observe part of the liver but fixing the liver to make the image more stable is problematic. In this experiment, a special vacuum suction cup is used to fix the liver and obtain a more stable image of the liver under the microscope. In addition, this method can be used to observe dynamic changes of specific substances in the liver by marking such substances with dyes.

In this experiment, a mouse is injected in its tail vein with Rhodamine B isothiocyanate&#8211;dextran that can stain blood vessels. After the liver is exposed and fixed, a specific part of the liver can be selected to observe the deep tissue in the living body using multiphoton microscopy.

老鼠肝脏组织血管多光子微观观测

Observing the intravascular dynamics of mouse liver tissue allows us to conduct further in-depth observations and studies on tissue-related diseases of ...

生物工程

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

<meta charset="utf8"></head><body>小鼠肺移植中细胞动力学的实时监测

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>作者聚焦：通过活体成像推进肺移植免疫学

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

移植小鼠肺的活体双光子显微镜检查

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...