Name: real-time imaging of myeloid cells dynamics in apcmin/+ intestinal tumors by spinning disk confocal microscopy
Uploaded: 2014-10-06T14:00:00.000+00:00
Duration: 5 min 21 s
Description: Watch this Scientific Journal Video about Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy at JoVE.com

我们要感谢英于APC的最小/ +小鼠基因分型。这项研究是由来自INSERM基金和美国国立卫生研究院资助（CA057621和AI053194）的支持。

注:所有的动物实验均按照批准的机构动物照顾与使用委员会（IACUC），加州大学旧金山分校的程序进行。所有成像实验是非生存过程和动物立即安乐死图像采集结束后。 1代小鼠注:APC 最小/ +小鼠，携带突变的APC基因上，自发形成50-100腺瘤在小肠。 <ol><li>横装甲运兵车最小/ +小鼠与ACTB-ECFP线，其表达ECFP下，肌动蛋白启动子，并进一步与C-FMS-EGFP线，其表达EGFP下的c-fms的启动子来检测骨髓细胞。杂合子动物ACTB-ECFP和c-FMS-EGFP和用于检测荧光。 </li><li>用小鼠在3-4个月的婴儿图像清楚地发现腺瘤。 </li></ol> 2，准备的M材料板手术前注:在显微镜建立的细节描 ​​述6,7是以前。 <ol><li>显微镜<ol><li>打开加热毯。打开为488 nm激发和固态405纳米，561纳米和640纳米激光器的氩激光。打开显微镜，纺丝盘控制单元，所述声光可调滤波器，激光控制单元，相机控制器。 </li><li>打开显微镜快门，果然LED的红色。打开电脑和摄像头软件。 </li></ol></li><li>成像平台<ol><li>确保舞台的插入是干净的。否则，请用肥皂和水，然后擦干。 </li><li>举两个盖玻片，用实验室磁带，以确保他们的插件。将插入在舞台上，并用酒精擦拭清洁。 </li><li>使用实验室胶带，附加加热毯右侧的阶段。 </li><li>验证的橡胶隔膜的完整性上的鼻锥和如果necessar改变它年。位置和使用纱布和实验室磁带修复鼻锥的异氟醚麻醉分娩的动物。 </li></ol></li><li>异氟醚麻醉系统<ol><li>笔芯的异氟醚储罐。 </li><li>打开真空并调整到1.2升/分。 </li><li>加蒸馏水至雾化器和雾化器连接到异氟烷系统。 </li><li>打开氧气分析仪以及将其连接到异氟烷系统。打开氧气罐，并确保氧分析器表示100％。调节O 2流量0.2升/分钟。 </li><li>打开氮气罐和调节流量为0.8升/分。确保氧分析仪显示21％。 </li></ol></li><li>外科工具和平台的准备<ol><li>干净的2对解剖镊子和剪刀用肥皂和水。 </li><li>打开热珠灭菌并让它达到250℃。消毒的外科手术工具，持续30秒。让他们冷静下来。 </li><li>将实验室透雨手术板凳上。 </li><li>使用保丽龙盖的手术平台。盖上一块实验室透雨的。准备第二块实验室透雨的剃须后鼠标来改变。 </li><li>放置在鼻锥与麻醉有关的覆盖泡沫塑料盖的线，并将其与一些胶带附着到泡沫塑料盖（未在实验室中裂化反应室），并使用两针上的鼻锥的两侧，以将其固定到位。 </li><li>组装优碘，酒精擦拭，消毒纱布，强力胶，显微镜​​载玻片，电动剃须刀，4个实验室胶带。 </li></ol></li></ol> 3，制备注射剂<ol><li>引擎盖下，制备胰岛素注射器（28国祥½in）的7微升股票AF647标记的莱曼6G（Gr1的）抗体（1毫克/毫升）的入100μl的2000 kDa的罗丹明 - 葡聚糖（4毫克/毫升）在眼窝注射。眼窝注射，建议在装甲运兵车最小/ +小鼠由于贫血，在这些动物的发展，使尾静脉difficuLT检测通过皮肤。 </li><li>准备第二次胰岛素注射1毫克/公斤阿托品稀释成150μL的PBS皮下（SC）注射前成像挫伤肠道蠕动。 </li></ol> 4，准备在肠道的成像<ol><li>验证麻醉线到感应室是开放的，并且在线路的手术台和显微镜都关闭。 </li><li>动物转移到麻醉室。关闭麻醉室并打开异氟醚到4％（含21％O 2）。 </li><li>当鼠标深呼吸，并慢慢地（经过5-6分钟），将其传送到手术台上的侧翼和注入的Gr1抗体溶液和/或葡聚糖溶液眼眶。 </li><li>打开连接到外科平台的麻醉线。收线到麻醉室，把鼠标放在它与它的鼻子早在麻醉锥。 </li><li>减少异氟醚浓度从4％至2.5％。 </li><li>验证鼠标很好捏它的脚垫和测试角膜反射麻醉。 </li><li>添加眼科润滑膏对眼睛，以防止干燥时在麻醉下。 </li><li>加入实验室胶带固定鼠标的手术分期的四肢。 </li><li>通过使用电动剃刀除去从腹侧表面上的毛发。也从左侧后肢去除毛发以定位采集之前的血氧计。从手术分期取出实验室透雨，换上干净的，避免头发受到污染。 </li><li>消毒腹面用酒精湿巾和优碘。 </li><li>注射阿托品溶液（来自3.2）SC在小鼠中的一个侧面。 </li><li>请通过皮肤1厘米的腹正中切口及腹膜用剪刀和一个钳子。小心拉出3-4厘米小肠，并确定一个或多个肿瘤图像。 </li><li>乘坐玻璃MICRoscope需要滑动和位置就可以了肠道有肿瘤上方的循环。 </li><li>添加一些强力胶沿肠道几个地点将其附加到幻灯片。等待一分钟的胶水干。使用标记来画一条线在幻灯片上指向肿瘤，以促进其本土化的共焦。 </li></ol> 5，将鼠标在舞台上，并准备进行图像采集<ol><li>从四肢取出实验室胶带。 </li><li>打开麻醉线到显微镜载物台，并关闭所述一个到外科手术平台。 </li><li>仔细传输鼠标显微镜载物台，其腹面朝下，其在鼻锥鼻。安全与某些实验室带麻醉行。 </li><li>重新定位，以便在将盖子滑窗中的一个的中心的肠道的鼠标和/或滑动。轻轻大盘回落与实验室磁带的幻灯片。 </li><li>附测定仪的探针的鼠标的左肢到按照它的心脏和呼吸频率和血氧饱和度。 </li><li>涵盖了鼠标与加热毯，以避免体温过低。 </li><li>从2.5％降低异氟醚浓度为1-1.5％用于成像。 </li></ol> 6，图像采集利用μManager软件<ol><li>增加CSUX速度为2500 RPM，以提高在配置设置的图像质量。 </li><li>使用目标下拉菜单选择10X 0.5 NA或20X 0.75 NA Fluar透镜物镜。 </li><li>选择颜色下拉菜单中选择激光405纳米。 </li><li>点击Live和调整与显微镜的焦点。 </li><li>点击自动自动调节基于所述图像中的极端像素值的显示图像的最大和最小像素强度。显示图像的像素亮度直方图呈现在图表中的主窗口的下部。 </li><li>通过调整派珀控制面板窗口中的相机曝光改变时钟的数量（一个时钟是33.333毫秒）。 </li><li>如果该信号是太亮具有最低曝光，使用显微镜控制单元降低激光强度。 </li><li>检查信号强度为488纳米，561纳米和640纳米的激光线。调整激光强度与激光自身的控制单元（488纳米和561纳米）或显微镜控制单元。 </li><li>在多D采集窗口，检查时间点框，并输入时间点数目和所需的时间间隔。 </li><li>检查Z-堆栈中，选择"相对Z"和定义的Z开始和相对Z-端的当前位置。 注:为10X，20X或目标，除了分别开始3 Z平面，8微米或4微米。 </li><li>检查多个位置（XY）中成像的几个地点，然后单击编辑位置列表中，选中4至6个不同的位置。 </li><li>检查通道中，并添加来自新的渠道和选择激光从下拉菜单和典型线路E在曝光对于每个信道（在33.333毫秒的倍数）。 </li><li>检查影像保存在"多D采集"窗口。 </li><li>选择一个文件夹，所有采集的图像会被自动保存在这个文件夹中特定的名称前缀。 注:每个连续的收购将被保存在一个单独的子文件夹作为单独的TIFF文件中的每个颜色，每个时间点每个Z片。 </li><li>通过点击多D采集窗口保存设置保存所有设置。 </li><li>单击收购多维采集窗口，开始采集。 </li><li>如果测定仪的探针不使用或停止工作以及（当脉冲是不稳定或难以检测），通过夹持足垫和检查角膜反射检查麻醉的效率的摄像过程中，每隔15分钟。调整麻醉，如果鼠标响应这些刺激。 </li><li>采集后，通过提高异氟烷concent安乐死的小鼠配给到5％。如果没有呼吸动作检测，从舞台移开鼠标，并进行颈椎脱位。 </li></ol>图像利用了Imaris软件分析7。 <ol><li>转换到.ims文件（ 补充图1A） <ol><li>打开摄像头软件，点击文件，然后批量转换。 </li><li>点击添加文件，选择第一个位置的第一个文件。重复每个位置上。 </li><li>对于上传的每个文件，点击设置，验证设置"T"代表时间，"C"的通道和"z"为Z-堆栈中出现的顺序。 </li><li>保存在指定的文件夹。浏览并转化为创建一个新的文件。 </li><li>点击设置为所有和启动。 </li></ol></li><li>调整（ 补充图1B） <ol><li>进入具体的转换文件夹，打开的第一个文件。 </li><li>在显示调整窗口，调整背景通过在必要时修改的最小数目为每个信道的信号截止。 </li><li>如果需要的话，通过修改最大数目（下的最大数量，较高的信号的强度）调节的信号强度。 注:信号强度/对比度调整使细胞行为或隔间的亮点，才能有一个更好的可视化。它不改变结果本身。 </li><li>点击编辑和图像属性。 </li><li>改变的x，y和z的值（为一个10X物镜，x和y = 0.712，Z = 8微米;对于20X物镜:x和y = 0.36，Z = 4微米）。 </li><li>点击所有等距调整的时间点。添加的开始日期和开始采集时间。添加结束日期和结束收购（ 补充图1C）的时间。 </li><li>点击播放按钮观看影片。 </li><li>点击编辑和删除的时间点，删除图像模糊。 </li><li>加入两个单独收购峠疗法为一体的电影，去的第一部电影的最后一个时间点，然后点击编辑&gt;添加的时间点，添加第二个文件。 </li></ol></li><li>保存为电影（ 补充图1D） <ol><li>点击记录，选择.MOV延伸和0的压缩因子。 </li><li>单击Save（保存）。 </li></ol></li></ol>

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The authors have nothing to disclose.

在本文中，详细的协议描述中纺肠道肿瘤骨髓细胞动力学盘共聚焦成像几个小时的活的动物，从肠道的浆膜面成像。 避免炎症反应，有最佳的生理条件下，在肠道的摄像，必须在完整器官进行。然而，从肠的浆膜侧成像是具有挑战性的，由于光具有到达上皮之前要经过不同的组织层，例如平滑肌。着眼于上皮可以在某些方面是困难的，特别是在肌动蛋白-ECFP报告小鼠由于肌动...

大量证据现在表明，在肿瘤微环境，包括异质细胞群，包括成纤维细胞，内皮细胞，免疫和炎症细胞，细胞外基质，和可溶性因子，通过促进几乎所有起着起始和实体瘤的进展的关键作用癌症1标志。的确，在肿瘤进展，也有发展，以产生微环境有利于恶性2转化的肿瘤细胞和基质细胞之间恒定的动态相互作用。间的免疫细胞浸润的肿瘤微环境，骨髓细胞是最丰富3。肿瘤相关巨噬细胞中的组成（TAM），髓源抑制细胞（肌源性干细胞），树突状细胞（DC）和嗜中性粒细胞（中性粒细胞），骨髓细胞从骨髓中招募和渐进渗透的肿瘤，释放出细胞因子，生长因子和蛋白酶的可以促进肿瘤的生长和扩散4。肿瘤细胞和骨髓细胞之间的串扰是复杂的，而是动态的。因而它们之间的相互作用的性质的认识是用于确定为什么这些细胞促进癌症的进展，而不是参与抗肿瘤免疫应答的关键，并且可以有助于发现新的目标，以控制它。 直接观察活体显微镜提供了活体小鼠5的组织中细胞动力学信息。四色，多区域，微透镜式旋转盘共聚焦系统被设计成乳腺肿瘤6中学习基质细胞。这种方法使长期连续成像，并包括若干优点，诸如（a）快速图像获取，以减少运动伪影，（二）长期麻醉，（三）四种颜色的采集遵循不同的细胞类型，（四）的荧光标记不同肿瘤部件，和（e）观察的不同肿瘤微环境机智欣相同的鼠标，避免鼠标鼠标的变化7-9。通过该技术，不同的细胞行为已有报道在乳腺肿瘤病毒（MMTV）启动子驱动的多瘤病毒中间Ť癌基因（PyMT）模型，该模型显示肿瘤发生的渐进阶段。调节性T-淋巴细胞（Treg细胞，由Foxp3的EGFP转基因的可视化）优先在接近迁移到血管而树突（CD11c的-DTR-EGFP），癌相关成纤维细胞（FSP1 + / +-EGFP）和髓样细胞（C-fms的-EGFP）显示出更高的运动在肿瘤周边比肿瘤块内。急性全身缺氧的条件下，细胞迁移是不同的:停止调节性T细胞相反，骨髓细胞是继续往前6迁移。此外，在相同的小鼠模型中，已经显示与肿瘤阶段的多柔比星的敏感性的变化，药物的分布与药物的反应，以及多柔比星处理导致CCR2依赖招募骨髓细胞的肿瘤。因此，实时成像，也可用于深入了解药物反应的原位和耐药10,11的生物。 性腺瘤性息肉病（APC）基因突变通常发生在人类结直肠腺瘤和癌12的APC基因导致家族性腺瘤性息肉病（FAP），它赋予了极高的风险结肠癌13的一个副本变异。在小鼠品系装甲运兵车 最小 / +进行截断突变的APC基因的密码子850和自发发展多种肠道腺瘤各地小肠14-16。肠的长期活体成像是因为该过程的侵袭的具有挑战性的，因为在打开腹腔是必要的访问权限的肠。短期实时成像STudies先前已公布在健康肠17,18，但长期直接观察肠道肿瘤尚未见报道。一种外科手术已被设计和精制通过肠道的浆膜表面显现肿瘤，使用先前用于图像的乳腺肿瘤6,10的活体旋转盘显微镜系统。在本文中，一个协议描述了允许一个由通过 APC调节最小 / +小鼠跟随在小肠中的肿瘤内的髓样细胞的行为。

<table><tbody><tr><td>&lt;em&gt;Apc&lt;sup&gt;Min/+&lt;/sup&gt;&lt;/em&gt; mice</td><td>Jackson Laboratory</td><td>2020</td><td></td></tr><tr><td>ACTB-ECFP mice</td><td>Jackson Laboratory</td><td>3773</td><td></td></tr><tr><td>cfms-EGFP mice</td><td>Jackson Laboratory</td><td>18549</td><td></td></tr><tr><td>2,000 kDa Dextran, rhodamine-conjugated</td><td>Invitrogen</td><td>D7139</td><td></td></tr><tr><td>Isoflurane</td><td>Butler Animal Health Supply</td><td>29450</td><td></td></tr><tr><td>Nitrogen</td><td>UCSF</td><td></td><td></td></tr><tr><td>Oxygen</td><td>UCSF</td><td></td><td></td></tr><tr><td>1x PBS</td><td>UCSF cell culture facility</td><td></td><td></td></tr><tr><td>Saline Buffer</td><td>UCSF cell culture facility</td><td></td><td></td></tr><tr><td>Anti-mouse Ly-6G (GR1) antibody AF647</td><td>UCSF Monoclonal antibody core</td><td></td><td>Stock 1 mg/ml. Use at 7 &amp;mu;g/mouse</td></tr><tr><td>Atropine</td><td>LARC UCSF</td><td></td><td>Use at 1 mg/kg mouse</td></tr><tr><td>Alcohol wipes</td><td>Becton Dickinson</td><td>326895</td><td></td></tr><tr><td>28 G x 1/2 in. insulin syringe</td><td>Becton Dickinson</td><td>329465</td><td></td></tr><tr><td>Remium cover glass</td><td>Fisher Scientific</td><td>12-548-5M</td><td>24x50-1</td></tr><tr><td>Betadine</td><td>LARC UCSF</td><td></td><td></td></tr><tr><td>Heat blanket</td><td>Gaymar Industries</td><td></td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td>Turn ON 30 min before use</td></tr><tr><td>Cotton tipped apllicators 6-inch</td><td>Electron Microscopy Sciences</td><td>72310-10</td><td></td></tr><tr><td>Anesthesia system</td><td>Summit Anesthesia Support</td><td></td><td></td></tr><tr><td>Inverted microscope</td><td>Carl Zeiss Inc</td><td>Zeiss Axiovert 200M</td><td></td></tr><tr><td>Stage insert</td><td>Applied Sientific Instrumentation</td><td></td><td></td></tr><tr><td>Mouse Ox oximeter, software and sensors</td><td>Starr Life Sciences</td><td>MouseOx</td><td></td></tr><tr><td>Nebulizer</td><td>Summit Anesthesia Support</td><td></td><td></td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td></td></tr><tr><td>&amp;mu;Manager</td><td>Vale lab, UCSF</td><td></td><td>Open-source software</td></tr><tr><td>ICCD camera</td><td>Stanford Photonics</td><td>XR-Mega-10EX S-30</td><td></td></tr><tr><td>Spinning disk confocal sacan-head</td><td>Yokogawa Corporation</td><td>CSU-10b</td><td></td></tr></tbody></table>

real-time imaging of myeloid cells dynamics in apcmin/+ intestinal tumors by spinning disk confocal microscopy

髓系细胞是在肿瘤内最丰富的免疫细胞和已被证明能促进肿瘤进展。现代活体成像技术使观察脏器内部活细胞行为的，但可以在一些类型的癌症，由于器官和肿瘤的辅助具有挑战性，例如肠。直接观察肠道肿瘤以前尚未报道。这里描述的手术过程可直接观察活体小鼠肠道内的肿瘤骨髓细胞动力学的利用转基因荧光报告小鼠和注射示踪剂或抗体。为了这个目的，一台四色，多区域，微透镜式旋转盘共聚焦显微镜，允许长期连续成像的快速图像捕获已被使用。 装甲运兵车 最小 / +小鼠的发展在小肠多发性腺瘤是正交与C-FMS-EGFP小鼠可视化骨髓细胞和ACTB-ECFP老鼠以可视化的隐窝肠上皮细胞。用于标记不同肿瘤成分，如血管和嗜中性粒细胞，并通过浆膜表面定位的肿瘤进行成像的方法的程序也被描述。从几个小时影像编定时短片允许髓细胞行为的原位肠道微环境的分析。

通过使用旋转盘共聚焦显微镜，非肿瘤和肿瘤组织在小肠装甲运兵车最小/ +的; ACTB-ECFP;的c-FMS-ECFP小鼠可以从浆膜表面可视化。成像后，照相机软件用于分析和调整的采集（ 附图1）。静脉注射（IV）注射荧光2000 kDa的葡聚糖-罗丹明B和莱曼6G 647结合的抗体后，血管和中性粒细胞可分别检测到（ 图1）。淋巴集结是淋巴组织的聚集和全骨髓细胞可?...

Watch this Scientific Journal Video about Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy at JoVE.com

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

real-time-imaging-myeloid-cells-dynamics-apcmin-intestinal-tumors

Research

JoVE Journal

Immunology and Infection

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

9.6K 次观看.  INSERM U661, Functional Genomic Institute. By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model. 

视频: Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

科研

癌症研究

使用植入式稳定窗的小鼠胰腺成像

Stabilized Window for Intravital Imaging of the Murine Pancreas

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have high morbidity and mortality. Intravital imaging (IVI) is a powerful technique enabling the high-resolution imaging of tissues in both healthy and diseased states, allowing for real-time observation of cell dynamics. IVI of the murine pancreas presents significant challenges due to the deep visceral and compliant nature of the organ, which make it highly prone to damage and motion artifacts. 
Described here is the process of implantation of the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP). The SWIP allows IVI of the murine pancreas in normal healthy states, during the transformation from the healthy pancreas to acute pancreatitis induced by cerulein, and in malignant states such as pancreatic tumors. In conjunction with genetically labeled cells or the administration of fluorescent dyes, the SWIP enables the measurement of single-cell and subcellular dynamics (including single-cell and collective migration) as well as serial imaging of the same region of interest over multiple days. 
The ability to capture tumor cell migration is of particular importance as the primary cause of cancer-related mortality in PDAC is the overwhelming metastatic burden. Understanding the physiological dynamics of metastasis in PDAC is a critical unmet need and crucial for improving patient prognosis. Overall, the SWIP provides improved imaging stability and expands the application of IVI in the healthy pancreas and malignant pancreas diseases.

作者聚焦:通过先进的活体成像彻底改变胰腺疾病的理解 

We present a protocol for the surgical implantation of a stabilized indwelling optical window for subcellular-resolution imaging of the murine pancreas, allowing serial and longitudinal studies of the healthy and diseased pancreas.

用于小鼠胰腺活体成像的稳定窗口

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have ...

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 ...

免疫与感染

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 ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

医学

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

小鼠巨噬细胞吞噬的延时3D 成像

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

应用先进的背皮窗室对转基因荧光小鼠肿瘤相关血管的活体显微镜观察

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

在髓系细胞动态实时成像

摘要

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Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

用于小鼠胰腺活体成像的稳定窗口

用于小鼠胰腺活体成像的稳定窗口

用于小鼠胰腺活体成像的稳定窗口

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

成像的CD4 T细胞间质迁移发炎真皮

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

小鼠巨噬细胞吞噬的延时3D 成像

应用先进的背皮窗室对转基因荧光小鼠肿瘤相关血管的活体显微镜观察

毛里小肠内皮淋巴细胞的内质成像