This work was supported by the Deutsche Forschungsgemeinschaft (DU 1190/3-1).

所有的动物实验均遵照执行与德国动物保护法（TierSchG）。小鼠安置，并按照良好的动物的做法来处理由FELASA（www.felasa.eu/guidelines.php）和国家动物福利的身体GV-SOLAS（www.gv-solas.de/index.html）定义。弗莱堡大学的动物福利委员会以及地方当局（Regierungspräsidium弗赖堡）批准的所有动物实验。 1.动物处理，制备和炎症的感应<ol><li>使用4 - 6的周龄雄性小鼠的16重量范围 - 20克。注意:如果老鼠是老年​​人或较重他们目前周围血管过多的脂肪，限制了显微观察。 </li><li>所有消毒手术前的工具和显微镜下。 </li><li>注射20mg / kg的LPS（脂多糖）腹膜内4至镜检到活小鼠以诱导细菌inflamm事先小时通货膨胀。 </li><li> Prewarm在水浴0.9％盐水溶液在37℃下加湿的塑料腔室和肠系膜组织。 </li><li>麻醉小鼠腹腔注射氯胺酮（100毫克/千克）和赛拉嗪（5毫克/千克）的显微镜过程之前。替代麻醉可以使用， 例如，2％异氟醚吸入。确认正确通过麻醉响应反射性刺激（或趾掐尾以坚定的压力）的损失。 </li><li>用剃须刀脱毛腹部并取出松散的头发用纱布用70％乙醇饱和。 </li><li>适用于鼠标的眼睛兽医药膏，以防止干燥，而在麻醉下。 </li></ol> 2.手术<ol><li>消毒用70％的乙醇的腹部。此方法不是无菌的方法和可能是致命的在实验结束。 </li><li>执行中位数剖腹探查:用小的，弯曲的镊子和小剪刀打开腹部皮肤。确定外延gastrical船只和白线的区域打开腹膜，保护血管。 </li><li>申请几滴预热盐水进入腹腔，以保持组织湿润。 </li><li>标签白细胞和血小板的荧光通过注入50μl的罗丹明6G作为9,10-之前所述（1毫克/毫升）眼眶。 </li><li> Exteriorize ileumand地方它的环中，其直径为10cm的培养皿，并确保通过将37℃，以保持组织湿润预热生理盐水溶液（0.9％），每隔一分钟。 </li></ol> 3.活体显微镜<ol><li>放置鼠标在显微镜下，把肠系膜静脉的直径为200 - 在视中心300微米。选择没有看得见的脂肪周围的船只。 </li><li>请勿触摸肠系膜血管从而避免了血管内皮细胞的刺激。谨慎处理回肠循环。 </li><li>可视化血细胞内皮相互作用与一个倒置或直立显微镜和用显微镜软件的相机。记录血液中的每只小鼠4个不同的脉1分钟的细胞内皮的相互作用。 </li><li>完成成像实验安乐死后的小鼠颈椎脱位。 </li></ol> 4.分析<ol><li>分析离线贯彻和蒙蔽所有参数。使用任何合适的软件程序进行分析。 </li><li>确认稳定interindividually可比的血流状况高时间分辨率电影，短片（最大帧速率）专注于管腔内血细胞流动。 </li><li>量化轧制白细胞数。为此提请通过静脉的垂直线和计数越过这条线在1分钟内所有的白细胞。 </li><li>通过测量一个单一的白细胞需要传递的50微米的距离，而稳定的滚动内皮细胞上的时间确定滚动速度。要做到这一点，画两条垂直线在50微米吨的距离hrough静脉。 <ol><li>测量一个代表白细胞需要从一行除以50微米通过所需要的时间（微米/秒）以获得到other.Calculate白细胞的速度的时间。 </li></ol></li><li>测量白细胞粘附在0.04 mm 2的字段。要做到这一点，绘制边长为200微米到静脉广场。 <ol><li>算上牢固粘附白细胞，定义为不可见的运动进行30秒，这个正方形内。 </li></ol></li><li>计数血小板绑定到一个白细胞量化血小板白细胞相互作用的次数。 </li></ol>

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The authors have nothing to disclose.

在这里，我们描述了准备和小鼠体内的肠系膜静脉活体显微镜的性能。这种方法使我们有机会直接在活的有机体观察白细胞，内皮细胞和血小板内皮细胞相互作用11。 作为早期对炎症的反应内皮被激活，并与白细胞和血小板产生的滚动，粘附和轮回12相互作用。但白细胞也与血小板形成所谓的相互作用，血小板白细胞复合物，主要是血小板中性粒复合物（PNCS）和血小板单核配合物（两局）13,14。这些相互作用可以观察和量化在此方法。 另外，该技术允许调查内皮活化与像VCAM-1或ICAM-1的标记物（ 例如，血小板/白细胞相互作用后）15。 该过程本身的物理刺激引起一定程度的内皮细胞活化，这导致白细胞可见轧制（但几乎没有缓慢滚动，没有牢固的粘合）。这意味着考官需要处理非常谨慎的肠系膜。如果存在由粗引起处理它会导致升高的号码轧制和粘附白细胞过多活化。达到30可再现数目 - 80每分钟轧制白细胞在野生型小鼠审查员已被训练。 P-选择/ PSGL-相互作用的堵塞可以作为一个控制确认白细胞与内皮的相互作用。它排除了该白细胞被被动地"附加"到内皮，如果血流量大大减少可能发生的一个场景。 肠系膜血管直径范围从约200 - 300微米。因此，这种方法不适合于评估微循环中的毛细血管，但重点在小静脉发炎，白细胞轮回的主要场所白细胞 - 内皮相互作用。相比其他帧内活体显微镜方法如睾模型，这种方法是更容易学习，并且可以在较短的时间周期来执行。 一个该方法的最重要的限制是，小鼠需要年轻没有太多肠系膜脂肪有一个良好的观点进血管，所以，这种方法不能长期喂养或治疗研究之后进行。 一旦审查员被训练，不同炎性病症可以检查用这种方法，如应用的LPS，巯基乙酸，TNFα或组胺。特定的血细胞的更具体的成像可通过使用靶向荧光标记的分子来实现。总体而言，该方法是简单易学和一个快速的方法来检查在活生物体白细胞 - 内皮和血小板白细胞相互作用。

此协议的目的是描述在活的小鼠的炎症性条件下直接观察的白细胞 - 内皮和白细胞 - 血小板相互作用肠系膜静脉的活体显微镜的简化技术。 活体显微镜的开发是为了研究炎性病症1,2，这是不平凡的，但重要的是理解的炎性白细胞募集和作用下在体内白细胞-内皮相互作用。在这个协议中，我们描述了一种基于此前公布的出版物开发。类似于可视化血小板结合在越来越血栓3和平行于什么已经公布早4，形象化肠系膜静脉通过透射光显微镜检查。也有内部的重要显微镜等各种模式，如大鼠5或小鼠肝和大鼠6的提睾肌。肠系膜静脉的活体显微镜已经开发和由几组在以前的研究中的应用。我们已经用这种方法来观察在野生型小鼠和色氨酸hydroxylase1（TPH1）之间白细胞募集的差异 - / - 小鼠，这是不足型非神经元血清素和比较结果，以小鼠的血小板血清素药理学由长期贫应用的选择性血清素再摄取抑制剂氟西汀7。我们也研究白细胞-内皮相互作用后急性氟西汀治疗8。 在这个协议中，我们集中在肠系膜静脉的活体显微镜，因为该模型可迅速地进行，并允许白细胞 - 内皮和血小板白细胞相互作用测量值有效。这是更大的挑战和耗时在其他器官，如骨，肝，皮肤，或提睾肌的活体显微镜。该模式升这里描述是具有炎性刺激物，如腹腔内注射脂多糖challengeing后的理想的炎性细胞 - 细胞相互作用的重现性的评价。

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		<tr>
			<td>Rhodamine 6G</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;989-38-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli 055:B5</td>
			<td>Sigma-Aldrich</td>
			<td>L2637&#160;</td>
			<td></td>
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intravital microscopy of leukocyte-endothelial and platelet-leukocyte interactions in mesenterial veins in mice

活体显微镜是一种可用于研究在不同的区域在活的动物不同的工艺和容器的方法。在这个协议中，我们描述肠系膜静脉的活体显微镜。这可以在时间与重复的结果示出在体内白细胞-内皮相互作用短时间内进行。我们描述了内皮的LPS攻击后的炎性设置。但在此模型中可以应用许多不同类型的炎性病症，如细菌，化学或生物和调查药物的给药以及它们对活的动物直接影响及其对白细胞募集的影响。此协议已被成功地应用到了许多小鼠和它们对血管炎症反应的影响的不同处理。在此，我们描述了通过荧光罗丹明6G标记这些白细胞和血小板的可视化。另外，任何特定的成像可以是PErformed使用有针对性的荧光标记分子。

在这个协议中所描述的实验设置的概述示于图1，它显示了一个鼠标与形象化回肠环和其容器，其可以在活体显微镜进行观察。 图2显示了一定程度的活化在未治疗的动物由于该过程本身。但几乎不存在慢滚动或白细胞相比LPS处理的小鼠的牢固粘结。 图2还示出了活体显微镜在野生型小鼠无论是与氟西汀在饮用水或未经处理的小鼠治疗3周。 图2A-的不同的结果C示出没有任何进一步的炎症挑战与LPS的结果，而小鼠在图2F-H用LPS另外处理。在这里，我们可以看到更高的数字滚动和粘附白细胞，同时滚动速度LPS刺激后下降。通过这些实验，我们可以表明，五羟色胺血小板为crucial为白细胞募集的炎症7的初始步骤。 在图3中，而无需进一步LPS攻击，只是后腹膜内2小时在手术前用氟西汀急性治疗，进行活体显微镜。在这里我们可以看到更高的数字牢牢粘附白细胞和更低的滚动速度在氟西汀治疗的小鼠，表明急性治疗氟西汀对白细胞-内皮相互作用8的影响。 在图中的脂多糖诱导性腹膜炎4血小板白细胞相互作用静脉内显现。血小板玫瑰花结周围的白细胞和这些复合物与血管壁相互作用。 <img alt="图1" src="/files/ftp_upload/53077/53077fig1.jpg" /> 图中的实验设置1.概述。 </strong&gt;（A）的麻醉鼠标形象化回肠环路及其系膜血管（黑色箭头）。 （B）的活体显微镜放置鼠标（红色箭头）的目标（黑色箭头）下方。光源（黑色箭头）和相机（红色箭头）标记。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53077/53077fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53077/53077fig2.jpg" /> 图2.活体显微镜经过3周的管理氟西汀对野生型小鼠无论有或没有LPS挑战。从滚动白细胞7（A）号修改而不LPS刺激。 （ 二）白细胞速度没有LPS刺激。牢牢粘附白细胞无LPS刺激（C）的数量。（四）未经处理的WT鼠标无LPS刺激血管的例子。 （E）氟西汀治疗的小鼠没有LPS刺激血管的例子。滚动白细胞（F）数LPS刺激后。 （G）LPS刺激后白细胞速度。对LPS刺激后紧紧贴壁的白细胞（H）的数量。 （Ⅰ）的未处理的WT小鼠LPS攻击后的容器的实施例。氟西汀处理的小鼠LPS攻击后的容器（J）的实施例。比例尺= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53077/53077fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53077/53077fig3.jpg" /> 图3:活体显微镜后急性氟西汀挑战2小时之前手术出LPS挑战。从8的改进）滚动白细胞数目没有LPS刺激。 B）白细胞速度没有LPS刺激。牢牢粘附白细胞没有LPS刺激C）的数量。 D）未经处理的WT鼠标无LPS刺激血管的例子。 E）急性氟西汀治疗的小鼠没有LPS刺激血管的例子。比例尺= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53077/53077fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53077/53077fig4.jpg" /> 图4: 在体内的血小板白细胞相互作用 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53077/53077fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 血小板白细胞间代表图片期间脉脂多糖诱导性腹膜炎小鼠体内的行动。从11再版。

Watch this Scientific Journal Video about Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice at JoVE.com

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

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 ...

intravital-microscopy-leukocyte-endothelial-platelet-leukocyte

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.1K 次观看.  Heart Center, University of Freiburg. 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.

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

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. ...

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 ...

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, ...

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 ...

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. ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...