Name: long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window
Uploaded: 2016-10-06T15:00:00.000+00:00
Duration: 7 min 19 s
Description: Watch this Scientific Journal Video about Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window at JoVE.com

This research was supported by NIH-CA100324, Einstein National Cancer Institute's cancer center support grant P30CA013330, R01CA172451 to JWP and the Integrated Imaging Program. This technology was developed in the Gruss-Lipper Biophotonics Center and the Integrated Imaging Program at the Albert Einstein College of Medicine. We acknowledge the support of these Centers in this work. The authors thank Mike Rottenkolber, Ricardo Ibagon and Anthony Leggiadro of the Einstein machine shop for their skilled and timely craftsmanship, the laboratory of Matthew Krummel for generously sharing their window design drawings, Kevin Elicieri and Jeremy Bredfeldt for their expertise in microscopy and their amplifier recommendations and Allison Harney and Bojana Gligorijevic for informative discussions.

在本协议中所述的所有程序都按照为使用脊椎动物，包括医药机构动物护理和使用委员会的爱因斯坦医学院的事先批准的准则和条例被执行。 1.生成荧光标记的小鼠模型和肿瘤细胞<ol><li>通过将0.1g的BSA用100ml的PBS混合制备100ml的0.1％（重量/体积）牛血清白蛋白/磷酸盐缓冲盐水（BSA / PBS）缓冲液。 </li><li>通过稳定转染制备荧光标记的肿瘤细胞。 注意:在这里，我们使用E0771-LG的细胞，从C57BL / 6小鼠的肺开发转移性肿瘤中分离该E0771小鼠乳腺腺癌细胞37的高转移性衍生物与亲E0771细胞38静脉内注射。 <ol><li>转染前24小时，板材1×10的60毫米组织培养皿5 EO771-LG细胞上2毫升antibio的抽动自由10％FBS（胎牛血清）的DMEM（Dulbecco氏改良的Eagle培养基），并在37℃和5％的CO 2。 </li><li>在转染时，加入190微升减少血清的介质的前孵育荧光蛋白载体的2微克与10微升转染试剂的30分钟。 </li><li>洗涤EO771-LG一旦细胞用Dulbecco氏磷酸盐缓冲盐水（D-PBS），并轻轻地添加转染混合物。 </li><li>孵育在37℃，5％CO 2的6小时。 </li><li>洗转染的细胞和培养在2毫升完全DMEM（10％FBS，1mM的丙酮酸，100U / ml青霉素，100微克/ ml链霉素）2天。 </li><li>洗涤细胞用2ml无菌PBS，加入500μl0.25％胰蛋白酶-EDTA，并在37℃孵育2分钟。 </li><li>收集细胞悬液并用至少1体积完全DMEM的混合，并在280×g下离心。 </li><li>悬浮细胞在1毫升完全DMEM，扩大细胞培养成10cm的组织培养皿。 </li><li>通过加入700微克/毫升G418的选择性抗生素的8毫升完全DMEM和文化的一个星期，变换媒体每三天开始转染细胞的选择。 </li></ol></li><li>从转染的细胞已经通过荧光激活细胞分选（FACS）39,40一直下选择在G418的充实荧光人口。 <ol><li>洗涤细胞用5ml PBS中并加入1.5毫升的0.25％胰蛋白酶。 </li><li>孵育在37℃下2分钟，并以完全DMEM中的至少一个体积混合。 </li><li>收集细胞悬浮液，在280 XG降速并在1mL的无菌0.1％（重量/体积）BSA / PBS缓冲液重新悬浮细胞。 </li><li>通过40微米的筛网过滤器的细胞悬浮液，并调整体积至1ml用0.1％BSA / PBS缓冲液进行排序。 </li><li> 10％的人口亮基于使用流式细胞分选机41荧光光谱FACS排序。 </li><li>文化的排序细胞一个星期ü升气管选择（700微克/毫升G418的完全DMEM）中。 </li><li>通过流式细胞术再次重复步骤1.3.1至1.3.6重新选择荧光标记的细胞流动。 </li><li>如所描述（步骤1.3.1-1.3.4）的第二轮选择，trypsinize，过滤器和后重新悬浮在0.1％BSA / PBS的细胞。调整浓度至2×10 6个细胞/ ml的单细胞分选到使用FACS分拣机96孔板中。 </li><li> 3-5准备96孔板用100微升完全DMEM的收集。要排序后提高生存率，从那里EO771-LG细胞生长42菜肴加100微升过滤的培养基。 </li><li>将细胞分选到96孔板后，返回细胞培养2天（37℃，5％CO 2）。 </li><li>通过检查在5倍放大在倒置显微镜下识别与可行的单个克隆的孔中。 </li><li> Trypsinize和扩大可行的克隆，冷冻细胞进行备份。 <ol><li>用洗井克由于用100μl无菌PBS的克隆。添加0.25％w / v的胰蛋白酶50微升，并且在37℃下孵育2分钟。加入50μl完全DMEM的并转移到无菌的V形底或圆底96孔板在280 xg离心降速5分钟。 </li><li>悬浮细胞在100μl700微克/毫升G418的完全DMEM的和板在12孔板的每个克隆。加入400微升完全DMEM与G418和文化直到融合。 </li><li>洗孔用500μl的PBS中，添加100μl的0.25％胰蛋白酶并在37℃下孵育2分钟。加入100μl完全DMEM和降速在280×g的5分钟。重悬的细胞在100微升与在6孔板直到汇合G418和板细胞完全DMEM的。 </li><li>一旦汇合，trypsinize细胞，降速重悬在FBS 10％DMSO冷冻细胞股票。 </li><li>保持在培养的细胞悬浮液的1/10通过在100mm组织培养皿镀他们的转移潜能的评估。 </li></ol></li></ol></li><li>测试选择的克隆转移潜能。 <ol><li> Trypsinize并重新悬浮在盐水中的细胞以2.5×10 6个细胞/ ml的浓度，并通过尾静脉注射200微升到C57BL / 6小鼠静脉内。 </li><li> 2周后，收集肺组织，如前所述23和由表面计数或体视方法43量化肿瘤负荷。选择具有高转移潜能的活体成像的克隆。 </li></ol></li><li>提高MacBlue（髓系特异性启动子驱动青色荧光蛋白（CFP）的表达（CSF1R -GAL4VP16 / UAS-ECFP 16））记者44只。 注:通常，使用8至12周的小鼠，但早在7晚20星期过测试工作，以及老鼠。 </li></ol> 2.多光子显微镜设置和成像准备注:虽然可以在任何m处执行这个协议ultiphoton显微镜，用于获取在这个协议中所示的数据的系统进行了详细45如前所述。 <ol><li>打开所有显微镜和激光组件，包括双光子激光器和检测器前方的期望的成像时间至少一小时。 </li><li>刚好手术前，通过只是在显微镜之前放置光功率计的头部中的光束路径测量的光输入到显微镜的功率，并调整激光端腔镜旋钮直到在880纳米的最大的光强度被读出上光功率计。 </li></ol> 3，真空系统设置<ol><li>胶盖玻片到成像窗口（参考图1）与腈基丙烯酸酯。允许至少4小时的胶水完全干透。 注意:此步骤也可完成的时间提前。 </li><li>切100μl的移液管尖端在与小开口的第一行和未切割的一端连接到薄vacuum软管。 </li><li>根据图1和补充图2连接的真空系统。 </li><li>用在真空和开放端受阻，调整为&lt;3英寸汞柱的真空调节器。 注:真空度的最终调整，将通过体内脉管的观察来进行的。 </li><li>申请石油油脂的薄膜的成像板，以防止物镜浸没介质从由成像板被邪恶远的下侧。 </li><li>放置在显微镜舞台成像板。 注:定制成像板（ 参考图3）是1/8的片"厚的铝加工既适合在阶段插入空间并在中心的通孔，用于保持所述成像窗口。 </li><li>插入所述真空窗口与盖玻片向下成像板。 </li><li>消毒所有表面和仪器，包括成像舞台板，和成像双赢陶氏用70％的乙醇。 </li><li>连接枪头到真空窗口的切割端和胶带将软管向下到成像板。 </li><li>使物镜靠近成像窗口，并将客观和盖玻片之间的大滴的水。 </li><li>保证有通过阻断真空窗口的中心开口并验证目标和盖玻片之间水滴不会被吸出从盖玻片没有泄漏。 注:放置在窗户的老式电脑鼠标球是堵塞中央开口有用。 </li></ol> 4.手术<ol><li>准备无菌手术区。 <ol><li>将所有仪器近在咫尺。消毒所有表面和仪器，包括手术区，并用70％乙醇外科手术工具。 </li></ol></li><li>领带2-0缝合3英寸长的导管，¼英寸带双结套以上。 </li><li>准备尾静脉导管FOL由哞哞叫哈尼等 46公布的协议。 </li><li>使用红外（IR）加热灯，温暖的动物在笼子里〜5分钟，以增加尾静脉血流，并在导管插入帮助。建议保持动物温热至生理温度在整个使用一个加热灯或变暖垫手术过程。 </li><li>麻醉用5％异氟醚的动物和验证是否有一个脚趾捏没有反应。 </li><li>适用眼药膏动物的眼睛。 </li><li>申请脱毛洗剂为10-30秒以从动物的左侧移除毛发;从胸部的中线到后面的四分之一，并从腋下到略低于胸腔。 </li><li>清洁任何多余的洗剂和消毒用70％酒精暴露的皮肤。 </li><li>附加填充有PBS中至尾静脉导管的无菌注射器，并代替继哈尼等人公布的协议插入和磁带。 </em&gt; 46。确保胶带被牢固地附着在针本身并没有在尾部和带之间的间隙自由。 </li><li>插管鼠标以下或者通过Das等 47或由杜佩奇等人公布的协议。48 </li><li>打开呼吸机，并设置成每分钟呼吸135和200微升异氟醚氧混合物。 </li><li>气管导管连接到呼吸机。 </li><li>将鼠标移动到手术区。要格外小心不打跑导管。 </li><li>领带的门牙周围下鼠标的鼻子2-0缝线。 </li><li>胶带将导管吻。 </li><li>大盘的左前肢的导管，以保持它的手术野。 </li><li>降低异氟醚麻醉2.5％的原有水平，并确认有一个脚趾捏没有反应。 </li><li>使用锋利的剪刀，拆下左胸壁皮肤上方1厘米2。 </li><li>电梯ŧ他乳腺脂肪垫和烧灼任何裸露的血管有烧灼笔。 </li><li>用锋利的剪刀切除脂肪垫。 </li><li>通过用锋利的剪刀切割除去肌肉层向下到肋笼。注意不要切腋静脉运行在前肢的基地。 </li><li>使用镊子抓住并提起第 6肋骨。使用以浅角度保持的锋利的剪刀（〜5°）切割肋邻近皮肤的开口的边缘。要格外小心，不要触摸暴露的肺组织。 </li><li>加宽在胸壁的开口通过除去四个连续的肋，以暴露整个肺叶。 注意:从胸骨保持开口至少5mm，以避免心脏。 </li><li>小心地抓住尾巴和气管导管解除鼠标和鼠标移动到显微镜成像阶段。 </li><li>与真空断开，填充用PBS真空窗口的腔室。 </li><li>反转鼠标和定位曝光肺在真空成像窗口。 </li><li>在真空慢慢转用球阀汞约3-5英寸。 </li><li>将在取得一半以上的鼠标和磁带到舞台板的胸前两次纸巾的限制安全带所示参考图2。 </li><li>剪辑脉搏血氧仪的大腿传感器动物的大腿上，并启动该软件。 </li><li>在舞台上放置的环境室并打开热能来维持鼠标在生理温度。 </li><li>减少异氟醚的水平1-1.5％维持麻醉，维持血流。 </li></ol> 5.活体成像<ol><li>使25×0.95数值孔径（NA）的物镜靠近盖玻片并在它们之间添加一大滴的水。 </li><li>使用表面荧光模式，查看FITC通道，并把肺组织成为关注的焦点。 </li><li>如果没有前做完手术，我nject肿瘤细胞通过尾静脉导管。 <ol><li>断开尾静脉导管PBS注射器。 </li><li>装载用100μl的肿瘤细胞悬浮液（2×10 7个细胞/ ml在PBS中的最大值）的无菌注射器。 注:该步骤可预先进行研究在不同时间点肿瘤细胞到达肺。 </li><li>与肿瘤细胞的注射器连接到尾静脉导管。 </li><li>缓缓注入肿瘤细胞到尾静脉。 </li><li>断开与从尾静脉导管的肿瘤细胞中的注射器。 </li><li>重新连接PBS注射器尾静脉导管。 注:注入葡聚糖当它变得难以通过注射后眼来区分从葡聚糖信号肿瘤细胞之前确定所有的肿瘤细胞的位置。 </li></ol></li><li>定位的肿瘤细胞将图像<ol><li>成像个别肿瘤细胞，找到所有的肿瘤细胞，并记录他们与日地点软件电子多点面板。 <ol><li>通过显微镜观察眼肿瘤细胞找到全部视野图像。 </li><li>在软件中，通过点击多点按钮切换到多点面板，并通过点击添加位置按钮存储单元的位置。 </li></ol></li><li>对于镶嵌影像，找到镶嵌的起源和设置成像坐标<ol><li>定位在结构的左上角的位置上被捕获。 </li><li>零通过按压阶段控制器上的"零"键台上的x，y和z坐标。 </li><li>通过点击"Load"按钮，然后选择列表加载了马赛克坐标相应的列表。 注:对于一个2×2马赛克的视图500μm的场的20％的重叠的例子清单将是:POS.1 =（0,0），位置。 2 =（400,0），波什。 3 =（0,400），波什。 4 =（400,400）。 </li></ol></li></ol></li><li>取下注射器W¯¯第i个的PBS在尾静脉导管和替换用含有葡聚糖的注射器。 </li><li>缓缓注入高达100微升的20毫克/毫升155 kDa的罗丹明 - 葡聚糖经尾静脉导管溶解在PBS入小鼠，随后通过注射50μl的无菌PBS冲洗线。不引入任何气泡成线。必要时，注入葡聚糖癌细胞注射后至少一小时，使施用至小鼠的总体积不超过4毫升/千克/小时。 </li><li>设置成像参数。 <ol><li>切换显微镜多光子模式。 </li><li>通过点击定时信号按钮，更新缩放系数字段设置变焦2倍的一个因素。 </li><li>调整激光功率，以〜10％（〜在样品10-15毫瓦）通过点击探测器和激光按钮，然后调整海啸电源滑块10。 </li></ol></li><li>图像中的每个位置，以验证肿瘤细胞的存在和可视化的vascu流动和完整性lature。 注:船舶用流动的红细胞出现充分灌注和荧光葡聚糖应该包含在血管内无渗漏到血管外空间。约10 - 20肿瘤细胞有望成为真空窗口的透明孔径内。 </li><li>调整每个位置的起始深度要成像。 <ol><li>对于每个位置，调整由阶段控制器上的调焦旋钮旋转到图像的肿瘤细胞的顶片的z轴位置。 </li><li>细胞中的视场的中心的位置。 </li><li>点击多点按钮，点击视野的位置以突出显示它，然后单击添加随后删除按钮来代替多点列表中的单元格的存储位置。 </li><li>目视观察在每个位置上的肿瘤细胞的相对亮度。 </li></ol></li><li>通过点击在多点面板的保存按钮保存每个小区的新的位置d指定文件名。 </li><li>对于个别肿瘤细胞成像，挑选大约相当于亮度的三个单元，并通过点击自己的位置，在列表中，然后点击删除按钮删除多点列表中的所有其它位置。 </li><li>点击探测器和激光按钮和调整滑块为绿色和红色通道45这样的光电倍增管的增益，该信号低于饱和。 </li><li>调整滑块蓝色通道45，巨噬细胞出现青色。 注意:任何二次谐波信号将只出现在蓝色通道，并且可以从青色的巨噬细胞通过以下信道减法过程来分离先前所述45。 </li><li>设置Z堆栈开始深度为0微米和最终深度为24μm分别移动Z阶段的位置并点击开始和结束按钮。 注:此深度范围内的细胞将具有最佳信号而被可视化噪声和分辨率。 </li><li>的Z步长设置为3微米。 </li><li>设置以下参数的摄像参数先前描述45,49。 <ol><li>对于个别肿瘤细胞的影像，点击定时信号按钮，然后输入4 V输入缩放系数场（相当于1.5倍的变焦因子），输入3到帧的平均场，然后点击时间推移按钮，输入10入定时字段。 注意:这些设置将收购1帧每3秒。 </li><li>对于镶嵌影像，点击定时信号按钮，输入1.5V的缩放因子（相当于4倍的变焦系数），帧平均数输入3，然后点击时间推移按钮，然后输入10到时间经过时间延迟场。 注意:这些设置将收购1帧每3秒。 </li></ol></li><li>启用通过点击其按钮多点，Z堆栈和T-推移成像模式。 </li><li>按录制按钮来获取图像。 不E:肺组织非常娇嫩，容易受到光损伤。如果在成像场的时间推移成像血流停止后，该激光是最有可能过高和随后的其它领域的成像必须在较低的功率来完成。 </li><li>每30-45分钟，慢慢注入50μl的PBS或生理盐水，以保持动物的水合。 </li></ol> 6.安乐死<ol><li>增加异氟烷％至5％。 </li><li>保持5％异氟醚下的动物，直到30秒它停止呼吸，从舞台上拆下后的动物。 </li><li>执行颈椎脱位，以确保完全安乐死。 </li></ol> 7.图像分析<ol><li>对于单细胞成像实验: <ol><li>将图像载入斐济和其格式为Hyperstack。 </li><li>在Hyperstack每个Z-片，玩的时间间隔电影，寻找残留的XY移动。如果发现残留的XY移动，适用叫StackReg 36插件在堆栈中，消除运动。 </li></ol></li><li>对于镶嵌成像实验: </li><li>将图像载入斐济和通过打开马赛克拼接宏（补充代码文件马赛克拼接），并输入信息有关的图像，如目录，文件基本名称，X和Y字段的镶嵌数和人数它们拼接片和时间点。 注:由于Java的如何解释目录，文件夹名称必须有两个反斜线作为分隔符的子文件夹。由于内置插件成对拼接的限制，基本文件名称不得包含任何破折号。 </li><li>为了获得脉管系统的界限的清晰视图，所有血液通道的时间点的平均值连成一个单一的形象，然后复制这个图像为背景的电影中其他渠道的每一帧。 注:这是通过简单的运行进行血液平均宏观完成（补充代码文件进行血液平均）。 </li></o升&gt;

The authors have nothing to disclose.

体内光学成像用荧光标记的功能标记，如蛋白质和抗体结合高分辨率急剧增加了转移级联的理解。它使直接可视化和单细胞，并在肿瘤细胞中的亚细胞参数，宿主细胞及其微环境的定量。原发性肿瘤内这种成像已经导致，例如，到离散的微环境是支持或者生长侵袭或传播6,7的发现。在入侵的情况下， 在体内成像揭示巨噬细胞和肿瘤细胞的共的迁徙流的在血管内7,50优...

高分辨率活光学成像已被证明是对理解许多生物过程，允许单细胞和亚细胞的参数被测量和量化的关键。在癌症研究，肿瘤和基质细胞的活体成像导致许多微环境相互作用6-11是仅在完整的动物本的发现。 关于体内单细胞分辨率的光学成像血管内和肿瘤细胞的传播在乳腺癌相关的微环境的发现甚至导致对预后和治疗反应的乳腺癌患者12-16新颖标记。适用于完整的内部重要器官的深处观赏的最佳成像技术是临床模式（MRI，PET，CT），它提供整个器官的美丽风景，并可以揭示病理它们产生临床症状之前也。他们无法，HH但是，揭示转移的驱动肿瘤进展的早期阶段和细胞机制由于缺乏单细胞分辨率。到时候肺转移，在这些模式可见，他们很好地建立和增殖。中的估计，90％的该到达肺要么不生存17或最初保持休眠18，它们比以前预期19，成像到达和生存的最早步骤更早到达播散的肿瘤细胞，并观察变得至关重要理解转移性接种和肿瘤生长的复发在偏远地点的过程。 表演肺这些意见已被证明不过非常困难;绝大部分影像学已经利用体外或植制剂20-23，只给个说法成单个时间点的肺。虽然这些准备工作确实提供了有用的信息细则第十五，它们不给微环境的各种部件之间发生的相互作用，因果关系，以及动态特性的完整的理解。缺乏适当的循环系统（与稳态伴随失调），并从人体的免疫系统的其余部分断开的使得它渴望来验证这些制剂在体内完整的组织产生的结论。 许多团体都进行了完整的肺2,4,5,24-33与Wearn和德国是第一个手术暴露胸膜层24和特里率先利用植入式成像窗口25的活体成像。 在肺高分辨率成像是由肺的恒定运动大大阻碍和几种技术已经开发了克服这种限制。瓦格纳和菲利浦27所研究的犬肺部的自然运动并设计了手术方案在一个相对固定的区域找到他们的植入窗口，而瓦格纳在他的窗口手术准备利用真空固定组织28。自那时以来，各种技术已被用于图像肺包括:支气管夹紧，顺序呼吸暂停和选通成像，过取样的采集，肺叶和真空34的胶合。每一种都有其优点和缺点，没有一种技术已成为优于其他34。例如，支气管夹紧和连续呼吸暂停改变气体在肺正常交流，并可能导致肺不张。门控成像和过采样收购不从这些缺点，但需要高速或专门的影像设备不普及。最后肺既胶合和真空技术避免两者上述缺点的，但是可能表现出剪切力引起的损伤，如果服务不是德恩。近年来，真空窗口已被小型化，并适于在使用共聚焦和多光子显微镜4,5,33和优异的高分辨率成像的小鼠用已经达到2。 表1概括了本丰富的历史，并强调那些描述新颖的论文进步在利用活体肺显像窗口。 本协议描述在现场，完整的肺使用较长时间推移多光子活体显微镜的图像转移具有最高分辨率亚细胞可能。图像使用配备有高数值孔径物镜和多个光电倍增管（PMT）探测器多光子显微镜获得长达12小时。转基因小鼠模型被用于荧光标记天然的巨噬细胞用荧光高分子葡聚糖和荧光蛋白转染的肿瘤细胞（标记脉管和肿瘤细胞respectivel沿Y）。虽然这种选择荧光标记的细胞使肿瘤细胞，内皮细胞 - 巨噬细胞的相互作用和动态可视化，该协议将荧光或无荧光，鼠标的任何应变工作。采集后，残余漂移运动（如果有的话）是使用斐济插件35,36和自定义宏时间平均血管通道，以消除由未标记的循环血细胞闪烁引起的消除。 虽然该协议的重点成像转移，该技术适用于观察到的与在肺高分辨率单细胞成像的许多其它生物过程。

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Catheters; 22 g x 1 in.&amp;nbsp;</td><td>Exel International</td><td>26746</td><td>Tracheal Catheter</td></tr><tr><td>PERMA-HAND Black Braided Silk Sutures, ETHICON LIGAPAK Dispensing Reel Size 2-0</td><td>VWR</td><td>95056-992</td><td>String</td></tr><tr><td>Liquid Super Glue, Clear, 0.14 oz</td><td>Hendel Corp.</td><td>LOC1647358</td><td>Cyano-acrylate Glue</td></tr><tr><td>Tetramethylrhodamine isothiocyanate&amp;ndash;Dextran</td><td>Sigma-Aldrich</td><td>T1287-500MG</td><td>155 kD Dextran</td></tr><tr><td>Laboratory Clear Tygon PVC Tubing, 1/16&quot; ID, 1/8&quot; OD, 1/32&quot; Wall Thickness, 25 ft. Length</td><td>McMaster Carr</td><td>5155T12</td><td>Thin Tubing &amp;amp; Tubing for Luer</td></tr><tr><td>Crack-Resistant Polyethylene Tubing, 1/8&quot; ID, 1/4&quot; OD, 1/16&quot; Wall Thickness, White, 50 ft. Length&amp;nbsp;</td><td>McMaster Carr</td><td>5181K24&amp;nbsp;</td><td>Thick Tubing</td></tr><tr><td>Depillatory Lotion</td><td>Nair</td><td>-</td><td></td></tr><tr><td>Micro Medical Tubing 95 Durometer LDPE</td><td>Scientific Commodities Inc.</td><td>BB31695-PE/1</td><td>Tubing for tail vein catheter</td></tr><tr><td>30 G x 1 in. BD PrecisionGlide Needle</td><td>BD</td><td>305128</td><td>Needles for tail vein catheter</td></tr><tr><td>Puritan Nonsterile Cotton-Tipped Swabs&amp;nbsp;</td><td>Fisher Scientific</td><td>867WCNOGLUE</td><td></td></tr><tr><td>Clear Polycarbonate Barbed Tube Fitting, Reducing Straight for 3/32&quot; x 1/16&quot; Tube ID</td><td>McMaster Carr</td><td>5117k51</td><td>Connectors between tubes</td></tr><tr><td>One-Hole Rubber Stoppers</td><td>Fisher Scientific</td><td>14-135F</td><td>Stopper for Vacuum Flask</td></tr><tr><td>SHARP Precision Barrier Tips, For P-100, 100 &amp;micro;l</td><td>Denville Scientific Inc.</td><td>P1125</td><td>Pipette Tip</td></tr><tr><td>Laboratory tape</td><td>Fisher Scientific</td><td>159015R</td><td></td></tr><tr><td>Puralube</td><td>Henry Schein Animal Health</td><td>008897</td><td>Opthalmic Ointment</td></tr><tr><td>Gemini Cautery Kit</td><td>Harvard Apparatus</td><td>726067</td><td>Cautery Pen</td></tr><tr><td>Graefe Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4&quot; Length</td><td>Roboz Surgical</td><td>RS-5135&amp;nbsp;</td><td>Forceps</td></tr><tr><td>Extra Fine Micro Dissecting Scissors 4&quot; Straight Sharp/Sharp 24 mm</td><td>Roboz Surgical</td><td>RS-5912</td><td>Sharp Scissors</td></tr><tr><td>Micro Dissecting Scissors 4&quot; Straight Blunt/Blunt</td><td>Roboz Surgical</td><td>RS-5980</td><td>Blunt Scissors</td></tr><tr><td>Wipes</td><td>Fisher Scientific</td><td>06-666-A&amp;nbsp;</td><td>Harness</td></tr><tr><td>PhysioSuite System</td><td>Kent Scientific</td><td>PhysioSuite</td><td>Vitals Monitor</td></tr><tr><td>1 ml Syringe, Tuberculin Slip Tip</td><td>BD</td><td>309659</td><td>Syringe</td></tr><tr><td>Cyano acrylate</td><td>Staples</td><td>LOC1647358</td><td>Cover Slip Adhesive</td></tr><tr><td>Petroleum Jelly</td><td>Fisher Scientific</td><td>19-086291</td><td>Water Barrier</td></tr><tr><td>Adapter Luer Cannulla 1.5 - 2.2 mm</td><td>Harvard Apparatus</td><td>734118</td><td>Catheter Connector</td></tr><tr><td>PhysioSuite MouseSTAT pulse oximeter</td><td>Kent Scientific</td><td></td><td>Pulse Oximeter</td></tr><tr><td>Isoethesia (isoflurane)</td><td>Henry Schein Animal Health</td><td>50033</td><td>250 ml</td></tr><tr><td>Oxygen</td><td>TechAir</td><td>OX TM</td><td></td></tr><tr><td>1x PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>PVC Ball Valve, Push to Connect, 1/4 In</td><td>Grainger</td><td>3CGJ7</td><td>Vacuum Valve</td></tr><tr><td>Small Animal Ventilator</td><td>Harvard Apparatus</td><td>683</td><td>Alternative is available from Kent Scientific: MouseVent</td></tr><tr><td>OptiMEM Reduced Serum Medium</td><td>ThermoFisher Scientific</td><td>31985062&amp;nbsp;</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>ThermoFisher Scientific</td><td>11668019</td><td></td></tr><tr><td>MacBlue Tg(Csf1r*-GAL4/VP16,UAS-ECFP)1Hume/J Mice</td><td>Jackson Laboratory</td><td>026051&amp;nbsp;</td><td></td></tr><tr><td>Multiphoton Microscope</td><td>Olympus</td><td>Fluoview FV1000</td><td>Alternative to custom built scope</td></tr><tr><td>Environmental Enclosure</td><td>Precision Plastics</td><td>Chamber for FV1000</td><td>Alternative to custom built enclosure</td></tr><tr><td>Phosphate Buffered Saline</td><td>ThermoFisher Scientific</td><td>14190136</td><td></td></tr><tr><td>Laser Power Meter</td><td>Coherent</td><td>FieldMaxIITOP</td><td></td></tr><tr><td>Laser Power Meter Head</td><td>Coherent</td><td>PM10</td><td></td></tr><tr><td>pcDNA3-Clover Fluorescent Protein Vector</td><td>Addgene</td><td>40259</td><td></td></tr><tr><td>G418 Sulfate Selective Antibiotic</td><td>ThermoFisher Scientific</td><td>10131027</td><td></td></tr><tr><td>MoFlo Fluorescent-Activate Cell Sorter&amp;nbsp;</td><td>Beckman Coulter</td><td>XDP</td><td></td></tr><tr><td>Trypsin EDTA 1x</td><td>Corning</td><td>25-052-Cl</td><td></td></tr><tr><td>40 &amp;micro;m Mesh</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>96 Well Plate</td><td>Costar</td><td>3599</td><td></td></tr><tr><td>60 mm Culture Dish</td><td>Corning</td><td>430196</td><td></td></tr><tr><td>10 cm Culture Dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A4503</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline 1x</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>C57BL/6J Mouse</td><td>Jackson Laboratory</td><td>000664&amp;nbsp;</td><td></td></tr><tr><td>Kim Wipes</td><td>Fisher Scientific</td><td>06-666-A&amp;nbsp;</td><td></td></tr></tbody></table>

long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window

转移到辅助站点，如肺，肝，骨，死亡率约90％1创伤性事件。这些网站，肺是最困难的使用活体光学成像评估由于身体，细腻的性质和维持适当的生理至关重要的作用范围内的封闭位置。而临床形式（正电子发射断层扫描（PET），磁共振成像（MRI）和计算机断层扫描（CT））能够提供该组织的非侵入性的图像，它们缺乏必要的可视化的最早播种事件分辨率，与单个像素由近千细胞。肺转移播种假设当前模型只是一个肿瘤细胞的到来之后发生的事件是确定性的生存和随后的生长。这意味着，与单细胞分辨率的2实时活体成像工具，以限定播种CEL的表型是必需ls和测试这些模型。虽然已使用各种体外制剂进行肺部的高分辨率光学成像，这些实验一般是单个时间点测定法和易受工件和可能的错误的结论，由于显着地改变的环境（温度，丛生，细胞因子， 等等。 ）从胸腔和循环系统3去除导致。最近的工作然而使用一真空稳定成像窗2,4,5中所示的完整的肺的那个时间推移活光学成像是可能的，典型的成像时间被限制在约6小时。在这里，我们描述了使用这种窗口历时12小时的肺进行长期活时间推移成像的协议。用这种方法得到的隔时图像的序列使在肺可视化和细胞 - 细胞相互作用的定量，膜动力学和血管灌注。我们进一步Ðescribe的图像处理技术，让肺微血管的空前明确的说法。

为了证明可以用这种方法可以实现的结果的类型，我们注射标记有荧光蛋白三叶草成MacBlue小鼠44的尾静脉在手术前不同时间点E0771-LG的肿瘤细胞。手术后，155 kD的罗丹明标记的葡聚糖注射IV标记进行血管和时间推移成像。 当成像的小鼠后24小时注射，单细胞是血管内可见，与巨噬细胞和单核细胞相互作用。这种情况的一...

Watch this Scientific Journal Video about Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window at JoVE.com

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

长期高分辨率活体显微镜在肺与真空稳定成像窗口

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

long-term-high-resolution-intravital-microscopy-lung-with-vacuum

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

13.1K 次观看.  Albert Einstein College of Medicine. This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window. 

视频: Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

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

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

免疫与感染

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 ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

研究癌症转移至肺部的永久窗口

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

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 ...

医学

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

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

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

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

使用真空稳定成像系统的实验性急性肺损伤中肺微循环的活体内宽视场荧光显微镜

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

<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 ...

长期高分辨率活体显微镜在肺与真空稳定成像窗口

摘要

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此视频中的章节

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

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

通过透明硅胶窗进行纵向活体成像

研究癌症转移至肺部的永久窗口

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

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

使用真空稳定成像系统的实验性急性肺损伤中肺微循环的活体内宽视场荧光显微镜

移植小鼠肺的活体双光子显微镜检查

移植小鼠肺的活体双光子显微镜检查

移植小鼠肺的活体双光子显微镜检查