我们感谢R. HEINTZMANN的富有成果的讨论的条带照明的方法，K. Rajewsky和A.哈伯曼的发展过程中提供的C57BL / 6 B1-8 GFP转基因小鼠。我们承认在授予NI1167/3-1德意志研究联合会（到RN），HA5354/4-1和SFB633，项目A15（至AEH），下批拉结 - 赫希奖学金的查理特（到RN）的资金支持。我们特别感谢网络JIMI进行富有成效的讨论及基础设施支援

多波束设置的条纹照明TPLSM 这里所使用的安装程序是一个专门的多波束双光子激光扫描显微镜，如先前所描述的6,20。该系统示于图1，本方法可以应用到其它的双光子激光扫描显微镜，其能够与本galvoscanner反射镜的运动同步相机采集，即使它们只能够执行单光束扫描。在这种情况下，缺点将是较低的采集速度，但是类似于标准的PMT基于TPLSM采集速度。为了达到最佳的品质，只要分辨率和对比度而言，以最低的光漂白和光损伤和最快的收购，但是建议考虑以下调整步骤设置的： <ol><li>检查蒂萨激光器的激光功率：检查达到100％，与原厂的价值观和PREVIO比较我们自己的读数，并使用它，因为它是，如果在范围700-1,000 nm的激发波长是必要的。 </li><li>测试远程控制指示为钛：萨光束分离器， 即检查为0％和100％的设定值。如果有剩余TISA束100％，然后检查并重新设置步进电机零位。在Ti：萨分束器包括一个低阶λ/ 2板和偏振片，这样会将在垂直于电子束路径的垂直偏振的激光束：一种被引导到显微镜激发，另一种是用于泵浦光参量振荡器（OPO）。自动旋转的λ/ 2板，在Ti的连续调节：Sa和OPO功率，分别实现。 </li><li>调节OPO到所需的波长，并测量输出功率。如果功率太低（与4 W钛宝石激光器在800 nm处，应该能够得到0.8-1 OPO光束W在大约1,050-1,100 nm）的优化泵波长和检查日的设置E镜的扇形水晶和。选择他们推荐的最佳值，通常可作为图表或曲线图，然后进行微调，直到OPO的共鸣在所需的波长和输出功率的增加。 <ol type="i"><li>如果电源仍然偏低，调节OPO的两个入口后视镜带四个访问镜面调节旋钮。做到这一步非常缓慢，在非常小的移动，前部和端部的镜子之间交替。 </li></ol></li><li>检查的Ti：Sa和/或OPO光束打在进入镜子在适当的中央位置。 <ol type="i"><li>用一张白纸（不是很有光泽或反射！）和一个红外查看器观察OPO梁和较高的波长的Ti：萨横梁。 <ol type="i"><li>用近红外钛时请戴上防护眼镜：萨梁（一般可达720-750 nm的可见对于一般人）。 </li></ol></li><li>设置激光功率（S）尽可能低的对准过程，以便宓nimize潜在的眼损伤。 </li><li>始终保持室内的环境灯，让眼睛的瞳孔收缩。 </li><li>请记住，激光功率是精细控制的衰减器组成了一个λ/ 2板和偏光板，这是不是在尚未生效时钛：萨光束进入扫描头！ </li></ol></li><li>检查入口点隔膜是由钛砸在了中间：Sa和/或OPO光束;如果不是，请调整最后两个反射镜将激光束进入扫描头之前。 <ol type="i"><li>使用IR观众和一小片白色的（但没有光泽/反光！）的纸张。 </li><li>总是在进入显微镜关闭隔膜，使得激光束的位置可以更精确地判断。重要提示：不要忘记进行步骤6之前，重新打开隔膜！ </li></ol></li><li>调整反射的激光束的光路进入显微镜相应。做到这一点的小片段，并执行迭代梁娃lking。 <ol type="i"><li>检查整个光路如果从物镜的光输出是不足够的。 </li><li>始终使用该系统的第一次很长的空闲周期，大修，更换任何光学元件，后当检查光路或者如果问题被怀疑与输出存在。 </li></ol></li><li>确保所有的主动光学表面的清洁，尤其是，他们是免费的灰尘！如果该表面覆盖有一层灰尘，波前被连之前进入物镜畸变和所产生的激光束将不会提供样品的鲜明的图像！ <ol type="i"><li>如有必要，请清洁后视镜带一张叠起来的镜头纸，蘸有乙醇或异丙醇。 </li></ol></li><li>检查光束到达背面到位于正上方的条目镜的棱镜型脉冲压缩器的端部，光路被设定后的镜子。从这里，该光束被反射的我NTO光束多路器（BM）。 </li><li>检查，如果激光束击中了位于所述光束多路器的入口，直接在λ/ 2板之前，改变激光束的偏振，以适应一个宽频带的50％透射和50％的反射的要求隔膜内BM。 <ol type="i"><li>首先，关闭光圈，以使光束位置可以更精确地判断和执行波束行走的两个膜片之间， 即 ，一个在进入显微镜和其他在BM条目。对于其他的单光束扫描显微镜隔膜应设在进入XY扫描器。 </li><li>如果光束不会碰到已关闭的孔的中心，调整上述反射镜，直接对BM条目隔膜之前放置。重要提示：不要忘记进行到第10步之前，重新打开隔膜。 </li></ol></li><li>与隔膜重新打开时，检查激光束击中BM镜子他们的中间点。用一张狭长地带，以免堵塞的BM的复杂的光路另一部分！ </li><li>检查，如果光束照射的出射反射镜的中间：顶镜，用于平行极化“P”，底部反射镜为垂直偏振的“S”。如果不是，在BM之前调整入镜。 </li><li>检查，如果“S”和“P”极化光束让组输入偏振器立方体和重叠适当地在进入XY扫描器。跳过的情况下，这些步骤的单光束扫描显微镜使用。 </li><li>检查该激光通过集中扫描和管透镜，并得到通过物镜的回焦平面的中间。重要的是：这个步骤必须用激光束在中心位置进行，而不是在一个停车位置！ <ol type="i"><li>与瞄准工具（“靶心”）更换物镜，检查激光束击中目标的中间，以及是否照度均匀。 </li><li>如果这看起来是这样，关闭光圈兽王之前，检查是否一个圆形的干涉图案出现时，用黑色圆圈中间（干扰最小）。 <ol type="i"><li>调整进入镜子，如果有一个显著转变相对靶心的中心。 </li></ol></li></ol></li><li>现在，随着物镜， 例如用20倍水浸泡镜片更换靶心。 <ol type="i"><li>检查输出光场用一张白纸。这应该是明亮，均匀，位置居中。 </li><li>测量输出功率：100％OPO 100％抽钛萨（4瓦），一个应该得到约在1100 nm处的透镜（在这里所使用的显微镜​​）后100-150毫瓦。较低的激光功率并不足以用于执行多波束SI-TPLSM。对于单束扫描SI-TPLSM 5-10毫瓦就足够了。激光束必须保持在中心位置而不被scanneD！ </li></ol></li><li>如果执行MB-SI-TPLSM，对准镜子中的BM如下： <ol type="i"><li>放置一个均匀荧光样品， 例如红色荧光幻灯片，在舞台上和焦点内的样品，但附近的顶面（ 即靠近物镜&#39;前）的激光束。 </li><li>显微镜设置为多光束模式和选择波束的数目，在一开始，优选只有一个光束，而极化， 例如 “P”。 <ol><li>发现激光束成像的荧光幻灯片：设置“P”快门打开并持续而聚焦光束运行的CCD相机。 </li><li>确保光束是在中锋位置上，并且扫描仪是关闭的。确保束未扫描！ </li><li>通过查找附近的摄像头芯片的中心亮点查找横梁。 </li><li>聚焦光束，直到光斑最亮和最清晰的， 即图像平面显微镜对应于相机芯片的位置;降低激光功率，以使光点不饱和。具有良好的对齐系统，这意味着激光功率在一个光束模式（大约0.6毫瓦的激光功率应该工作）工作时，靠近最低限度。 </li><li>如果点是显著偏离中心，将其移动接近中心通过调节反射镜前面的BM（或扫描仪的单束扫描）。 </li><li>当也对准其他两极分化， 即 “S”梁，现在切换到2束模式下，打开“S”快门和检查光束的位置（只有一个光束，如果只有“S”快门打开可见）。 </li><li>完成后，切换到4束模式，并使用“P”极化模式（“P”快门打开）。现在，2个子束就会出现。 </li><li>安排两个子束被完全平行于相机的视图的底部边缘，并将它们设置为2.8微米分开。 <ol type="i"&gt; <li>如果它们没有被正确对齐，调节BM的第一反射镜。 </li><li>从未对准螺钉在中间，而不是使用2周螺丝来移动反射镜，直到两个光束是2.8微米分开和完全水平地布置。 </li></ol></li><li>现在切换到8束模式中的“P”偏振，并调整第二反射镜BM（也无红点），直到4子束是等距于2.8微米，并且平行于图像的下边缘。 </li><li>重复16 - 和32 - 束模式，调整第三和第四BS镜，分别。为子束，因为SI-算法，最简单的最小 - 最大（希洛）算法，是基于这样的假设是等距离是很重要的。 </li></ol></li></ol></li><li>具有不均匀结构化的， 如交叉染色铃兰根茎代替均匀荧光样品。 <ol type="i"><li>去激发光束的停车位置，瓦特HICH通常放置在扫描仪坐标， 如 （10,000; 10,000）的较大值。 </li><li>扫描（多波束）的图像与CCD摄像头。检查图像清晰，均匀，平直。 </li><li>调整相机如果出现图像旋转：用手扭动机身轴向绕垂直轴（温柔！），直到图像拉直。 </li></ol></li><li>开始在xy扫描器，以控制扫描过程，以产生激发的模式， 即条纹的图像。 <ol type="i"><li>在多光束扫描的情况下，移动的Y扫描镜， 即得 。垂直于该子束线，以这样的方式来生成一个二次条纹图像。 </li><li>如果视场中生成如在步骤17.1中描述。太小时，通过移动与x扫描镜的位置确保该光束松懈线加倍和子束是沿着整个线等距离延伸的扫描程序。重复的精确运动的Y扫描镜后的X扫描镜被移动，以产生较大的条纹图像。 </li><li>在单光束扫描的情况下，反复交替用X扫描镜运动的Y扫描镜运动在等距离的位置 - 由科学家确定 - 来生成所需的有条纹的图像。 </li><li>确保使用用于Y扫描镜的命令为恒定速度（降低加速度/减速度）的运动和用于X扫描反射镜1在最大速度（最大加速度/减速度）。 </li></ol></li><li>自动获取和保存与相机（现场检测）的条纹图像。因此，同步摄像头，扫描仪，并使用扫描仪作为主触发。 </li><li>重复采集的条纹图像通过移动在不同位置的X扫描镜如下（以协议17所述的扫描协议）： <ol type="i"><li>选择足够的重复步骤，并与相应的步长随后的两个条纹图像，以覆盖两个连续的子束（在本例2.8微米，扫描仪反射镜1步等于25纳米）之间的距离;它可能是可取的，可以有少量的重叠， 即一个附加0.3-0.4微米。 </li><li>设置x移位到一个值，该值相匹配的横向分辨率限制在给定的波长。作为一个例子，使用10个步骤，每个移位和12或13的位移的X扫描反射镜的引导，以最好的轴向和横向分辨率的结果在本系统中。请与结构化幻灯片， 如铃兰根幻灯片，这数字在正在使用的系统效果最好。 </li><li>优化这两个值，直到铃兰图像变得最清晰：使用线轮廓工具所计算的SI图像和比较的谱线轮廓（它是从铃兰细胞壁的荧光信号）的宽度。 </li><li>收购与扫描仪的运动同步的每个条纹的图像并将其单独保存， 如 </em&gt;的TIFF或二进制文件。 </li></ol></li><li>打开一套完整的条纹图像为矩阵， 即 3D矩阵，在评估程序并使用最小-最大算法或傅立叶变换算法生成高分辨率的SI-图像的二维矩阵。该算法进行了详细的20如前所述。 </li></ol>小鼠免疫和准备活体成像动物实验已获动物福利（LAGeSo，Landesamt献给Gesundheit UND Soziales，柏林）适当的状态委员会，并根据目前的指导方针和法规（动物实验许可证G0153/08）被执行。 <ol start="21"><li>如4之前所述进行中的腘淋巴结和制剂的摄像视场的生发中心反应的诱导。纯化的B细胞免疫磁性从脾脏B1-8 + / +Jκ - / -和B1-8 + / +Jκ的B细胞- / -的GFP +小鼠。 </li><li>静脉内注射用的&gt; 95％使用⅛B1-8 + / +Jκ的纯度为3×10 6的NP特异性B细胞- / -的GFP +和⅞非荧光B1-8 + / +Jκ - / -在C57BL / 6的收件人。 </li><li>细胞移植后的一天免疫收件人与10微克NP-CGG（硝基苯鸡ɣ球蛋白），右后的食物垫在乳化完全弗氏佐剂（CFA）的。 </li><li> anti-CD21/CD35抗体ATTO590-琥珀酰亚胺基酯的标签Fab片段。 <ol type="i"><li>以突出滤泡树突状细胞的网络（FDC） 的体内生发中心内，注入10荧光物质标记的Fab片段微克到小鼠12-24小时的右后肢足垫进行活体分析之前。 </li></ol></li></ol>下面的步骤应conside红色为腘窝淋巴结的活体成像（8-10天免疫后）的准备： <ol start="25"><li>通过腹腔注射氯胺酮/赛拉嗪的，这取决于它们的重量的量麻醉小鼠。 </li><li>测试反射来监测麻醉深度在整个摄像期间。 </li><li>刮腿，背部和鼠标严谨的右侧，并用脱毛霜，以彻底去除残留的头发。 </li><li>修复右粗隆重要：找到骨片，用手指，设置一个小的皮肤切口，用剪刀，直到一个白色的小三角形筋​​膜出现。 </li><li>骨钳芯片这个筋膜与限制器的尖头镊子下方。 </li><li>修复脊椎使用齿镊子腰区。 </li><li>修复右后腿限制器并拧紧螺丝。 </li><li>带尾要保持腿部在正确的位置。 </li><li>在大腿的尾侧，建立在皮肤上的切口，在该区域中的突出腘静脉缓和RS的组织，从用生硬的镊子底层组织中分离皮肤，并按照腘静脉从近端到远端。 </li><li>用钝镊子暴露淋巴结，尽量避免切割，以保护优良的淋巴管（保持淋巴结在PBS中的暴露）。 </li><li>创建真空硅脂周围的淋巴结转了一圈，填补了这一水坑用PBS。 </li><li>把盖玻片在它的位置，然后将温度计探头（温度保持在37℃，否则电池的速度将显着不同）。 </li><li>沿盖玻片的上缘加的真空润滑脂的圆。 </li><li>把加热线圈在其位置上的真空润滑脂，和以前一样以同样的方式建立一个密封，并添加水/ PBS中于玻璃盖玻片蘸入目标。 </li><li>成像后，将小鼠保持在麻醉，同时增加剂量的氯胺酮/赛拉嗪的以致死剂量，随后通过颈脱位。 </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supraresolution+imaging+in+brain+slices+using+stimulated-emission+depletion+two-photon+laser+scanning+microscopy.">Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy.</a> Neuron. 63 (4), 429-437 (2009).</li><li>Bianchini, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-wavelength+two-photon+excitation+-+stimulated+emission+depletion+(SW2PE-STED)+super-resolution+imaging.">Single-wavelength two-photon excitation - stimulated emission depletion (SW2PE-STED) super-resolution imaging.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (17), 6390-6393 (2012).</li><li>Berning, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscopy+in+a+living+mouse+brain.">Nanoscopy in a living mouse brain.</a> Science. 335 (6068), 551 (2012).</li><li>York, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+doubling+in+live,+multicellular+organisms+via+multifocal+structured+illumination+microscopy.">Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy.</a> Nat. Methods. 9, 749-754 (2012).</li><li>Gu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advanced optical imaging. , (2001).</li><li>Cha, J. W., Ballesta, J., So, P. T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shack-Hartmann-wave+front-sensor-based+adaptive+optics+system+for+multi-photon+microscopy.">Shack-Hartmann-wave front-sensor-based adaptive optics system for multi-photon microscopy.</a> J. Biomed. Opt. 15 (4), (2010).</li><li>Booth, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+optics+in+microscopy.">Adaptive optics in microscopy.</a> Phil. Trans. R. Soc. A. 365, 2829-2843 (2007).</li><li>Niesner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+power+of+single+and+multibeam+two-photon+microscopy+for+high-resolution+and+high-speed+deep+tissue+and+intravital+imaging.">The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging.</a> Biophys. J. 93 (7), 2519-2529 (2007).</li><li>R&#252;ckel, M., Mack-Bucher, J. A., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+wave-front+correction+in+TPM+using+coherence-gated+wave-front+sensing.">Adaptive wave-front correction in TPM using coherence-gated wave-front sensing.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 17137-17142 (2006).</li><li>Tang, J., Germain, R. N., Cui, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superpenetration+optical+microscopy+by+iterative+multiphoton+adaptive+compensation+technique.">Superpenetration optical microscopy by iterative multiphoton adaptive compensation technique.</a> Proc. Natl. Acad. Sci. USA. 109 (22), 8434-8439 (2012).</li><li>Ji, N., Milkie, D. E., Betzig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+optics+via+pupil+segmentation+for+high+resolution+imaging+in+biological+tissues.">Adaptive optics via pupil segmentation for high resolution imaging in biological tissues.</a> Nat. Methods. 7, 141-147 (2009).</li><li>Lu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+laser+scanning+microscopy+through+spatiotemporal+modulation.">Super-resolution laser scanning microscopy through spatiotemporal modulation.</a> Nano Lett. 9 (11), 3883-3889 (2009).</li><li>Andresen, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+intravital+microscopy.">High-resolution intravital microscopy.</a> PLoS ONE. 7 (12), (2012).</li><li>Debarre, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+optics+for+structured-illumination.">Adaptive optics for structured-illumination.</a> Opt. Exp. 16 (13), 9290-9305 (2008).</li></ol>

沃尔克·安德烈森是LaVision生物技术有限公司，比勒费尔德，德国的股东。其他作者不存在任何利益冲突。

活体光学成像的目的是动态地和功能上可视化细胞运动和相互作用，以了解组织和在健康和疾病5器官功能。最有力的技术来实现这一点，多光子激光扫描显微术，仍然要克服与波前畸变，散射，缓慢采集，光漂白和光损伤，这限制了它的空间分辨率的限制，对比度以及其时间分辨率。 有两种成功的解决方案，以实现更好的激发（和发射）光子管理在深层组织导致更?...

双光子激光扫描显微镜（TPLSM），其优点为深层组织成像与红外超短脉冲激发1，已通过揭示各种运动和交互模式彻底改变了我们的看法在单细胞水平的生命过程细胞亚群在活体动物2-5。然而，目前的技术仍不足以澄清器官的功能和功能障碍的机制，作为开发新的治疗策略的先决条件，因为它使得大约只有在健康和疾病组织中的细胞反应的分子基础稀疏信息。当前技术使由于散射几百微米只有一个空间窗口和波上的空间分辨率和信号噪声比6，这是特别明显的成年动物的高度紧凑的组织面前的失真效应。这些散射和波前畸变的影响是由于高度异质的的折射率在组织三维各向异性分布，导致在第一步骤中，以三维空间分辨率的深度依赖性恶化，最后到信号从弹道激发光子， 即损失的信号-噪声比始发的总损耗。在生物科学和生物医学深层组织的应用程序而言，这意味着目前的技术是无法明确地揭示蜂窝通信，因为分辨率差，会导致假阳性的相互作用，而信号噪声比的减少将导致系统忽略了一些暗淡的结构之间的相互作用。 为了清楚地检测以动态方式的细胞相互作用，一个高度改善的空间分辨率，需要深的组织内。基于特殊的数值算法， 如结构照明方式，对第一激发态损耗， 如目前推出了功能强大的纳米显微技术</em&gt;受激发射损耗，RESOLFT，或在分子的定位， 例如 dSTORM，PALM，已经发现许多应用中固定的细胞，以及在活细胞培养7。然而，为了这些应用程序扩展到组织切片，活组织和有机体，我们仍然需要克服严重的技术困难。双光子激发的受激发射损耗具有不同波长，以及与单个波长（sw2PE-STED），用于激励和受激发射已被应用，以提高横向分辨率在大脑切片8或人工基质与包埋细胞9，分别在同一轴向分辨率为标准TPLSM。利用单光子受激发射损耗，树突棘的动态可以在大脑皮质（长达10-15微米的深度）的表面活Thy1系EGFP小鼠在67纳米10的分辨率成像。一个多功能的工具，用于发育生物学是由多灶性结构照明显微镜，它提供了两方面的改进二维提供分辨率。然而，这种技术可用于仅在与光散射如斑马鱼胚胎11倾向低的生物。不过，没有这些技术可以在成年动物的高散射组织被应用在数百微米，这是至关重要的模型与发病的疾病在出生后的生物医学和临床研究。 独立用于计算衍射限制的波前的形状， 即该点扩散函数（PSF）的近似值，聚焦通过透镜后，沿着光轴（轴向分辨率）的PSF的宽度大于至少3倍PSF的宽度在垂直于光轴（横向分辨率）12。波不同的订单面前的扭曲泽尔尼克的量化系数大大修改深层组织成像聚焦电磁波导致更大的PSF，特别是沿光纤的波前形状人轴13-15。因此，这两个衍射法和波前畸变效应指向沿着光轴在深层组织成像的限制因素的分辨率。而纳米显微技术专注于抵消衍射的极限只有一个技术，提高了轴向分辨率和通过抵消两个衍射和波前畸变效应的对比度是需要高分辨率的活体成像。理想情况下，这种技术应该也足够快，以便监测细胞动力学的。 PSF畸变和使用TPLSM自适应光学对比度损失的实时校正已被广泛研究，并在过去十年13,14,16-18改善，因此它是目前市面上最好的选择，从而更好地管理弹道导弹激励14的光子。不过，由于大部分波前校正方法在自适应光学系统中使用的其实是迭代和他们公顷已经被重复为小区域（数10×10微米2）由于在组织中的折射率的高异质性，采集速度比所需的成像细胞运动和通信显著更低。此外，在自适应光学的物理极限提高TPLSM仍然由衍射确定。 照明（SPIN）和检测侧（铲）时间调制的空间调制已经从理论上提出了要施加到激光扫描显微镜来提高分辨率。在活体成像的实际应用还有待证明19。 综上所述，对于技术，有效地提高了分辨率为深层组织成像在活成年动物发展的高需求。在这项工作中，我们实现了激励模式的，通过控制在多波束条带照明的多光子激光 - S的扫描过程空间调制罐头镜（MB-SI-MPLSM）20。相反，以结构化的照明方法，其中，所述激发光束的横截面进行空间调制，我们只使用扫描过程来实现激发的空间调制。通过扩大激发到更长的波长，我们是独立的光能够提高在高散射组织（ 如淋巴结，自由散射路径47微米6）深层组织双方的空间分辨率和信号噪声比，我们检测到的非线性信号， 例如荧光，产生二次谐波或其它混频现象。使用这种方法在激发波长为900毫微米，我们能够动态图像的细胞的蛋白质结构上的几个分子在小鼠淋巴结生发中心的规模。因此，我们可以更好地可视化在探测反过程滤泡树突细胞和B细胞的表面上的抗原携带单元之间的相互作用期间在次级淋巴器官的免疫应答根。

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">ATTO590 &#8211; NSH for coupling</td>
			<td style="width:99px;">ATTO Tec, Germany</td>
			<td style="width:119px;">AD-590-35</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">CD21/35 &#8211; Fab fragment &#8211; ATTO590</td>
			<td style="width:99px;">DRFZ, Berlin</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">The coupling reaction was performed in the central lab of our institution.</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">B1-8 Jk-/- EGFP+</td>
			<td style="width:99px;">Prof. Anne Habermann, Yale Univ., CA, US</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Can be also found at Jackson Laboratories (JAX).</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">EasySep Negative Selection Mouse B Cell Enrichment&#160;</td>
			<td style="width:99px;">StemmCell Technologies, Germany</td>
			<td style="width:119px;">19854</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Polystyren beads (605), 0.2 &#181;m</td>
			<td style="width:99px;">Life Technologies, Germany</td>
			<td style="width:119px;">F8803</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:99px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Name of Equipment</td>
			<td style="width:99px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">TriMScope I</td>
			<td style="width:99px;">LaVision Biotec, Bielefeld, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">OPO (manual version)</td>
			<td style="width:99px;">APE, Berlin, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Ti:Sa Laser Chameleon Ultra II</td>
			<td style="width:99px;">Coherent, Duisburg, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">water-immersion objective lens, 20x, NA 0.95, IR, WD 2 mm</td>
			<td style="width:99px;">Olympus Germany, Hamburg, Germany</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
	</tbody>
</table>

highly resolved intravital striped-illumination microscopy of germinal centers

监测蜂窝通信由活体深层组织的多光子显微镜的关键是理解厚组织标本和器官在健康和疾病中的免疫细胞的命运。通过控制多光子显微镜扫描模式和运用恰当的数值算法，我们开发了一个条纹的照明方式，使我们能够实现3倍更好的轴向分辨率和改进的信号噪声比， 即相反，在超过100μm的组织深度内高散射淋巴器官组织的标准相比，多光子显微镜。采集速度以及光漂白和光损伤作用类似于标准的光电倍增管为基础的技术，而成像深度是由于使用领域探测器略低。通过使用条带照明的方法，我们能够观察到的二次滤泡树突状细胞的免疫复合物沉积动力学̵1;上的几个蛋白质分子在生发中心的水平。

在淋巴结空间分辨率实际点扩展函数（EPSF）的尺寸对应于显微镜12的空间分辨率。我们通过我们的MB-SI-TPLSM获取在淋巴结胶原纤维的二次谐波生成的信号相比，建立了双光子激光扫描显微技术， 即场检测TPLSM（由CCD相机装置）测定该三维函数和点检测TPLSM（由光电倍增管的方式）。 从这些测量结果可以很明显地，在试件的表面，无论是横向和?...

Watch this Scientific Journal Video about Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers at JoVE.com

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

高分辨率的活体成像具有增强对比度高达120微米的深度在成年小鼠的淋巴结被空间调制多焦点双光子显微镜的激励模式来实现。在100微米的深度，我们测量了487 nm的分辨率（横向）和551纳米（轴向），从而绕过散射和衍射极限。

生发中心的高度已解决活体条纹照明显微镜

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

highly-resolved-intravital-striped-illumination-microscopy-germinal

Research

JoVE Journal

Immunology and Infection

9.9K 次观看.  Leibniz Institute. 高分辨率的活体成像具有增强对比度高达120微米的深度在成年小鼠的淋巴结被空间调制多焦点双光子显微镜的激励模式来实现。在100微米的深度，我们测量了487 nm的分辨率（横向）和551纳米（轴向），从而绕过散射和衍射极限。 

视频: Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Intravital Microscopy of the Inguinal Lymph Node

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

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

腹股沟淋巴结的活体显微镜

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

科研

免疫与感染

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

追踪中性粒细胞腔内的爬行，活体视频显微镜Transendothelial组织中的迁移和趋化

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

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

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

活体成像的鼠标使用2 - 光子显微镜的胸腺

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

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

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

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

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

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

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

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

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

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

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

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

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

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

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

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

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

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 ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

直立活体显微术制备小鼠颌下腺涎腺

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

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

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

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

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

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