我们要感谢这个手稿的严格审查，努诺莫雷诺博士为物流Foxp3的碘化钾GFP / GFP的实物捐赠有助于建立我们的动物持有人及电热垫和维杰-光Kuchroo博士大卫博士Olivieri小鼠。这项工作是由"Fundação第Ciência发送Tecnologia"（FCT，葡萄牙），授予＃PTDC/EBB-BIO/115514/2009支持。

1。动物准备重要说明:在无菌条件下进行骨髓细胞悬液和胸腺移植。骨髓悬液，我们的细胞培养室罩内，而胸腺移植是在位于我们的动物设施内的手术室进行。为了保持和保证这些无菌条件下，我们不允许我们的视频记录在这些地方。然而，所有的手术材料，灭菌和手术板凳以前用卫康消毒剂（医药品研究实验室公司）和70％的乙醇清洗。所有程序批准的机构动物护理和使用委员会（IACUC），他们在联邦与欧洲实验动物科学协会（FELASA）指令的协议。批准的ID号是AO10/2010。所有的图像实验终端程序和动物安乐死紧随图像年底收购。 详细过程如下: <ol><li>照射（与γ-辐照）8周龄裸小鼠900拉德内摧毁的骨髓（BM）的内源性造血前体。照射后，动物笼子，直到您准备的供体骨髓细胞静脉注射和进一步的骨髓重建。此骨髓转移后进行照射1小时。 </li><li>骨髓捐赠动物分开。一个动物的后肢股骨和胫骨一般都不够3收件人动物重组。与CO 2安乐死后，取出后肢，然后从骨子里清除皮肤和肌肉。 </li><li>剪下每个骨骼的封闭端，用PBS（或RPMI）冲洗或粉碎他们都在迫击炮，使用杵与2毫升PBS（或RPMI），删除的BM。 </li><li>扰乱蓬勃向上的愿望USI的重复的成单细胞悬液BM结构吴一个P1000的吸管。另外，您还可以通过整个21G注射器针头的BM几次。离心机（400G，5分钟，RT），暂停在PBS细胞，并注入你的裸体辐照受助静脉注射。在我们的实验中，我们使用BM - KI Foxp3 的GFP / GFP小鼠所有调节性T细胞（Treg细胞）表达绿色荧光蛋白 1 。 </li><li>接受捐助BM发生转移后的第一天，因为非BM重组动物无法存活超过14天。然而，应等待4至6周，让所有的BM车厢完全恢复。复方新诺明（罗氏）在照射后的第一周水（2毫克/毫升），以减少细菌感染的风险。 </li><li>胚胎胸腺移植已经描述2。简单地说，开始滋生的对等基因小鼠和堵塞（性交证据）每天检查。单独的插入女性和CO 2安乐死pregn 15天ancy（观察插头是怀孕的第1天）。取出胚胎及胸腺。直到转移放置在冷PBS胚胎胸腺。 </li><li>要执行的胸腺移植BM收件人，麻醉与氯胺酮（120微克/克鼠标重量）/甲苯噻嗪（鼠标重量的16微克/克）的裸鼠，并保持在37 ° C，直到他们得到一个加热垫的顶部昏迷不醒。 </li><li>手术暴露肾脏进行移植的胸腺。首先ChloraPrep（Carefusion公司）和70％的乙醇擦洗皮肤。然后，一个1厘米的背外侧的动物身体的一部分，2毫米波纹管的最后胸肋骨皮肤上切。 </li><li>切开腹腔，拉出本腔的肾脏。用手术刀刀片补肾胶囊，做一个小的浅表切。使用一个非常薄的尖钳，使肾囊下的收件人的鼠标一小袋，加起来这个袋子4胸腺叶。 </li><li>把肾b<html> ACK到它的地方，与一个或两针缝合腹腔，然后使用相同的缝合方法关闭小鼠皮肤。另外，缝合手术胶水可以使用。 </li><li>注入镇痛（布托啡诺在5毫克/千克）皮下注射后完成手术，以避免动物的痛苦，并保持在一个加热垫，直到他们开始恢复从麻醉中的动物。老鼠笼单独移植后第3天拆除缝线，以防止笼交配。取出1周后拆线。 </li><li>裸鼠没有T细胞。因此，您可以评估胸腺移植验收监测CD4 +或CD8 + T细胞在血液中的比例。每周一次，2周后胸腺移植，出血的动物和染色抗CD4和抗CD8单克隆抗体（单抗）的血液。当CD4:CD8比值约为2:1，移植的胸腺功能齐全（图1）。 </li></ol>"&gt; 2。活体图像采集准备好所有的材料，您需要提前，并把它们放在一边。 <ol><li>麻醉与氯胺酮/甲苯​​噻嗪（在项目1.5中所述的相同剂量）动物和它们放在一个加热垫的顶部在37 ° C。注入100罗丹明B异硫氰酸-葡聚糖（20毫克/毫升的PBS）静脉注射液的血流量，让未来的可视化。 </li><li>公开肾移植的胸腺，根据先前在项目1.6中所述的协议。 </li><li>为了防止肾脏返回到原来的位置，紧密缝合皮肤切口的侧面。 </li><li>放置一个PBS浸泡过的棉花片的器官，以保持湿润的顶部。 </li><li>将动物持有人阶段（图2A）顶部加热垫。 </li><li>把对动物的持有人，器官最多（图2B）在加热垫的顶部动物。 </li><li>捏住两侧钳位米的器官薄铝箔的ADE（图2C）。 </li><li>关闭动物的持有人及地方顶部加热器探头尽可能靠近你的组织（图2D）。这种热水器探头不断措施器官的温度，并将此信息发送到加热器调整电流，保持在37 ° C。 </li><li>取出PBS浸泡过的棉花，并在30 ° C准备顶部添加低熔点琼脂糖（2％）的PBS。 </li><li>盖顶部加热器（图2E）的准备。在此加热器的光圈有一个玻璃罩隔离琼脂糖从客观（图2F）。监视鼠标麻醉和注射半剂量升压，每40分钟。图像采集后，安乐死与CO 2的动物。 </li></ol> 注:铝箔剪辑和顶部加热器的图片图3 。 3。双光子成像收购我们用一个堂堂正正的"草原终极XY"双光子显微镜。我们的系统配备了钛:蓝宝石激光，四大光电倍增管同时进行多达4个通道的收购和20倍水浸泡的目的。 <ol><li>打开的钛蓝宝石激光和整个显微镜系统。 </li><li>添加分色镜和过滤器设置，根据所需的波长。在我们的例子中，我们使用奥林巴斯BX2色度持有五十〇分之五百二十五纳米过滤（Foxp3的GFP信号），和一个50分之595纳米过滤（葡聚糖 - 罗丹明- B的血管内的信号包含一个565纳米的分色镜， ）。使用激光的波长范围是880至900纳米之间。 </li><li>添加动物架显微镜，连接并打开加热器，加水顶部加热器光圈，放入水中仔细较低的目标，并重点对船只或一些GFP - Tregs的。 </li><li>用显微镜x，Y，Z外部控制，迅速调查的组织，以找到一个地区<html>利益。 </li><li>调整优化分色和最小化的实现足够的信号超过背景所需的激光量的光电倍增管的增益。尝试使用少量的激光，以避免您的组织的光损伤。 </li><li>一旦利益的地区是位于开始的时间推移成像。在我们的例子中，一个典型的5D（X，Y，Z，T，和颜色）的收购协议，由获得50微米深入组织体积，除以Z -步骤，X和Y的长度为140微米到4.0微米的连续图像每个。每卷30秒左右被收购。因此，60收购将执行图像采集约30分钟。 </li><li>将数据传输到体制服务器和使用ImageJ，Imaris，或Volocity软件进行多维呈现和细胞跟踪。 </li></ol> 4。代表性的成果<html> g1.jpg"/&gt; 图1。胚胎胸腺验收和功能的骨髓恢复后，胚胎胸腺移植骨髓再造动物;胸腺移植两周后，这些老鼠被放血每周一次的监测与抗CD4或抗CD8染色T细胞亚群的百分比单克隆抗体。我们认为胸腺是成功地在动物与CD4接受:CD8周围1.5-2.0:1的比例（一）。要确认它的功能，我们牺牲了一些胸腺移植的动物，并比较不同（二）WT小鼠胸腺细胞亚群的百分比。亚群（抗CD25和抗CD44单克隆抗体染色），双阳性（DP的CD4 + CD8 +胸腺细胞）;（ - - CD8 +胸腺细胞CD4 + DN）和单阳性（SP）的CD4 +双负的百分比+或CD8 +胸腺细胞之间的这些动物类似。 ig2.jpg"/&gt; 图2。动物持有人大会。深深地麻醉后，动物是加热垫之上，以前安装在顶部的动物持有阶段（一）。肾移植的胸腺是朝上（二）。铝箔夹微妙捏整个肾脏（三）保持它在的地方。图1C插入显示详细钳。 （四）地方动物持有人的上半部分。从顶部的器官，取而代之的是温暖的低熔化琼脂糖，PBS浸泡过的棉花被删除，并添加顶部加热器（五）。最后，整个大会转移到显微镜，这里的目标（F）的代表。 <img alt="图3" src="/files/ftp_upload/3504/3504fig3.jpg" /> 图3。持有部分的详细信息，这些图片展示的铝箔夹（一）持有肾（二）。还要注意在移植的胸腺是位于地区。这大约表明该地区削减胸腺囊，口袋，把胚胎胸腺。顶部加热器玻璃罩（三）固定用硅酮胶，其底部（d）部分。 电影1。Foxp3的- GFP +充氧和温度（37℃）的生理水平，在整个过程中保持胸腺内的胸腺细胞的活体成像。注血流量的调节性T细胞和运动。这些速度可测，并与公布的数据<a href="/files/ftp_upload/3504/3504movie1.avi">相比，点击这里查看</a>电影。 电影2。活体成像的调节性T细胞内收购胸腺图像30 ° C.注意运动和圆形细胞尽管维持血液流动的<a href="/files/ftp_upload/3504/3504movie2.avi">情况下，点击这里</a>查看电影。

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我们什么都没有透露。

在本文中，我们展示了一个活的动物胸腺细胞内双光子成像的程序。我们还介绍了一些参数，应仔细控制，如血流量的延续和器官的温度维持在成像过程。然而，尽管小心努力保持稳定的器官，如"器官漂流"的运动伪影，可发生。后路图像校正可以由专门为此目的而设计的算法的发展。进一步的图像分析，也可以是新的发展协议，旨在最大限度地减少错误的源。 胸腺器官，所有的T细胞产生的，因此，它是在兴趣了解γδ代的免疫学家在机关，CD4或CD8 + T细胞，将集中他们的注意力。大多数关于T细胞的研究是基于数字的差异和/或这些C稳定英语学习者后，在体外/体内操作的不同。然而，只有后在体内的可视化，我们可以观察到参与维持动态平衡 3-7免疫系统细胞之间的相互作用。因此， 在体内观察胸腺细胞可能是最重要的失踪信息，以便更好地了解T细胞生物学之一。活体TPM提供了T细胞的运动和相互作用的详细情况，我们将演示如何，它可以详细的胸腺细胞研究。然而，每个技术都有其局限性。虽然活体成像收购是反映身体内部的细胞行为的最准确的系统，它也是如此explanted器官的图像采集少费力，并已用于收集有关免疫系统8,9的重要信息。此外，一个不能否认活体成像方法需要暴露于麻醉动物组织和血管的手术，本身可能导致在整个器官的生理10变化。不过，也有非侵入性的方法， 取消 11外科手术新方法造成的文物正在开发，更好地提前做好准备要使用的 12只动物。因此，新的手术程​​序和收费，将最大限度地减少或绕过实际收购活体成像的局限性，并成为科学界越来越多的访问。 我们已经证明，我们所描述的方法是可行的， 它在体内系统性的操作，这类药品监督管理局报告所有，我们已经使用。因此，我们建议使用这种方法与已有的体外技术，以补充和加强有关胸腺细胞发展的进一步研究。

<table border="1"><thead><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr></thead><tbody><tr><td>若丹明B ishothiocyanate -葡聚糖</td><td> Sigma - Aldrich公司</td><td> R9379 </td><td>准备在20毫克/毫升的股票</td></tr><tr><td>双光子显微镜</td><td>草原科技公司</td><td>草原终极XY </td><td></td></tr><tr><td>钛蓝宝石激光器</td><td>相干公司</td><td>变色龙超家族</td><td></td></tr><tr><td> 20x/1.00 NA浸没目标</td><td>奥林巴斯公司</td><td> XLUMPLFLN 20XW </td><td></td></tr><tr><td>持有人（过滤器/分色） </td><td>色度科技股份有限公司</td><td> 91018 BX2（U型MF2） </td><td></td></tr><tr><td> 525 nm/50过滤器</td><td>色度科技股份有限公司</td><td> ET525/50m </td><td></td></tr><tr><td> 595 nm/50过滤器</td><td>色度科技股份有限公司</td><td> ET595/50m </td><td&gt; </td></tr><tr><td> 565 nm的分色</td><td>色度科技股份有限公司</td><td> 565dcxr </td><td></td></tr><tr><td> Imaris软件</td><td>位平面AG公司</td><td> Imaris </td><td></td></tr><tr><td> Volocity </td><td>珀金埃尔默公司</td><td> Volocity </td><td></td></tr><tr><td> ImageJ </td><td>美国国立卫生研究院， </td><td> ImageJ </td><td></td></tr></tbody></table>

intravital imaging of the mouse thymus using 2-photon microscopy

双光子显微镜（TPM），提供图像采集在深层组织和器官内的地区。结合新的立体定向工具和外科手术的发展，TPM成为一个强大的技术，以确定内部器官的"壁龛"，并记录在活的动物细胞的"行为"。虽然活体成像提供最类似于真正的内部器官的细胞行为的信息，这是比较费力，在技术上比其他前体内成像收购所需的设备/程序方面的要求。因此，我们描述了外科手术和小说"立体"，使我们能够遵循Foxp3的运动+细胞在胸腺器官持有。 Foxp3的调节性T细胞（Treg细胞）的产生是主控调节。此外，这些细胞可分为根据其原产地:即。胸腺分化调节性T细胞被称为"自然发生的调节性T细胞"（nTregs），为Opposed向外围转换调节性T细胞（pTregs）。虽然已在文献报道的关于这些T细胞表型和生理的研究量显著，很少有人知道他们在与其他细胞的体内相互作用。这种缺陷的原因可能是缺乏的技术，将允许这些意见。本文中所描述的协议提供了一个补救这种情况。 我们的协议包括使用，因为他们有一个准时在妥协一些上皮细胞，包括胸腺上皮细胞的分化的DNA序列突变，缺乏一种内源性的胸腺的裸鼠。裸鼠γ射线辐照和Foxp3的碘化钾GFP / GFP小鼠骨髓（BM）的重组。骨髓恢复（6周）后，每个动物胚胎胸腺移植的肾囊内。胸腺验收（6周）后，将动物麻醉;肾脏含有的移植的胸腺被暴露，固定在我们的机关持有人，并通过TPM的生理条件下保持体内成像。我们一直在使用这种方法，研究药物的调节性T细胞生成的影响。

Watch this Scientific Journal Video about Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy at JoVE.com

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

我们已经开发出新的实验室工具和胸腺的活体成像收购协议。我们的技术帮助识别"龛"胸腺内T细胞发育时。

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-imaging-of-the-mouse-thymus-using-2-photon-microscopy

Research

JoVE Journal

Immunology and Infection

16.9K 次观看.  Instituto Gulbenkian de Ciência. 我们已经开发出新的实验室工具和胸腺的活体成像收购协议。我们的技术帮助识别"龛"胸腺内T细胞发育时。

视频: Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

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

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 ...

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 Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

小鼠腘窝淋巴结的活体成像

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

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

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

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

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

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

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

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

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

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

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

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

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

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

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

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

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

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

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

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

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

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

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

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

使用IL-1β启动子驱动的红色荧光蛋白记者小鼠中性粒细胞引发的活体成像

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