This work was supported by EMBO (to Y.E.), Foundation Machaon (to Y.E.) and SNSF (to B.A.I.). We thank the Bioimaging Core Facility for the availability of the Nikon A1r microscope and technical assistance. We thank Mrs. Clarissa Bartley for English correction.

注：动物的程序是按照动物护理在瑞士日内瓦的机构伦理委员会和州兽医办公室进行。授权号GE / 63/14 1.准备一个单细胞悬液，从骨髓<ol><li>牺牲小鼠（8-12周龄）颈椎脱位。消毒后腿以70％的乙醇。从后腿去除皮肤。 </li><li>解剖出鼠标的股骨和胫骨和腿部用手术刀去除组织。冲洗的腿与90％的乙醇和地方变成培养皿填充有PBS。 </li><li>根据培养罩，干净的纸巾，从股骨和胫骨用手术刀和独立的膝关节。切断骨头的两端。 </li><li>从每个骨骼使用23G针和1ml注射器填充有制备培养基（无酚红的DMEM / F12，1％大鼠血清），到50毫升冲洗骨髓的螺旋管。通过经由23Gね微妙传代破坏细胞聚集体edle和1ml注射器。把总体积25毫升的准备中。 </li><li>离心机250 XG，5分钟，4℃。弃上清。在1ml重悬沉淀红细胞溶解RBLC缓冲液（8.3克氯化铵 ，1克碳酸氢钠，加入1ml EDTA（100毫米），完整到1L蒸馏水，过滤0.22微米）在15ml的螺旋管。孵育30秒在冰上。 </li><li>加入10毫升制备培养基和离心机250×g离心5分钟，4℃。弃上清，悬浮在2毫升的准备中。 </li></ol> 2.中性粒细胞富集阴性选择<ol><li>加入150微升鼠标中性粒细胞富集的鸡尾酒（细胞分离试剂盒的平台，如EasySep）。混合并在冰上孵育10分钟。 </li><li>加入10毫升酚红的DMEM。倒置一次和离心机250×g离心3分钟，4℃。 </li><li>弃上清。悬浮颗粒在1.75毫升制备介质在5毫升聚苯乙烯圆底管。加入250微升比奥在选择鸡尾酒。混合并在冰上孵育10分钟。 </li><li>涡磁性粒子（从细胞分离平台盒）30秒重悬颗粒。加入500μl磁性粒子的细胞悬浮液。混合并在冰上孵育10分钟。 </li><li>将管插入磁铁。等待3分钟。 </li><li>反转磁铁到一个新的5毫升聚苯乙烯圆底管，收集所需的未标记细胞。不要把最后一滴挂在管。不需要的细胞将保留在磁铁内部的管。丢弃该管成生物危险废物。 </li><li>将新管插入磁体。等待3分钟。 </li><li>反转磁铁到新款15毫升螺旋管，收集所需的未标记细胞。不要把最后一滴挂在管。 </li><li>完整体积至5ml用酚红的DMEM。 </li></ol> 3.标签中性粒细胞<ol><li>添加0.5微升橙色染料如细胞跟踪橙色（工作浓度为1μM，CMRA - 氯甲基rodol乙酸酯，库存的10mM在DMSO中）。在37℃下2分钟。 </li><li>加入2.5毫升的准备中。离心机250 XG，5分钟，4℃。 </li><li>弃上清。重悬沉淀在1ml制备介质在1.5ml管中。 </li><li>以等分用血细胞计数器计数。 </li><li>孵育5分钟，在37℃。离心机250 XG，5分钟，4℃。 </li><li>弃上清。重悬沉淀在1ml PBS中。离心机250×g离心5分钟，4℃下洗。 </li><li>弃上清。悬浮颗粒每100微升PBS 10×10 6个细胞 。置于冰上，直到静脉注射。注：电池必须IV尽快注射越好。 6-12x10 6中性粒细胞/鼠标，通常获得的。 </li></ol> 4.鼠标准备活体显微镜注意：步骤4.1）和4.2）应在实验开始时进行，以避免对IC标记的嗜中性粒细胞的延长的等待时间即<ol><li>称量CX 3 CR1 GFP小鼠（8-12周龄）。 </li><li>制备800微升麻醉药（氯胺酮60毫克/公斤，甲苯噻嗪4.5毫克/千克，acepromazone 1.75毫克/公斤的PBS）。 </li><li>麻醉鼠标。注射IP用200微升/ 20克鼠标。按捏脚趾并通过检查呼吸频率控制麻醉。 </li><li>注入的中性粒细胞（10×10 6个细胞在100μlPBS中）静脉内使用的侧面尾静脉或眶后窦，在实验者的便利性。 </li><li>把鼠标放在加热垫，保护眼睛，眼部凝胶。 </li><li>的玻璃盖玻片（直径为35毫米）放置在10厘米组织培养皿，它有一个直径30毫米的孔。使用油是维持组织培养皿中的盖玻片接触。 </li><li>切开腹部皮肤用剪刀，露出腹膜。腹膜切开用剪刀，露出肠。 </li><li>加入200μl的PBS（预加热，在37℃）到peritoneal腔。拿鼠标在你的手，把它面朝下，使肠道得到了腹腔中有一个温和的压力。鼠标放置在组织培养皿。 </li><li>轻轻deroll用消毒棉签将肠道到盖玻片，以暴露肠系膜血管。 </li><li>切纸组织的乐队，与37℃加热PBS弄湿。固定与面纸的部分小肠，以减少从肠蠕动而产生的运动。 </li><li>把组织培养皿和鼠标37℃恒温室内部到倒置显微镜的定做托盘阶段。引入含有麻醉药SC到后腿的注射器。注：此外，鼠标可以接收氧气（0.5升/分钟）用的掩模。 </li><li>控制鼠标麻醉通过脚趾捏和检查呼吸频率每30分钟。如果需要，提供麻醉药的提升（20微升）妥善维持麻醉。不E：应避免脉管的干燥。因此，它的湿度是通过定期润湿用于固定肠用37℃温热的PBS的纸巾保持。 </li></ol> 5.测量中性粒细胞和单核细胞行为发炎的血管在体内使用活体显微镜<ol><li>设置激光器488-和561-在需要避免激光诱导光毒性最低的功耗。在我们的实验中，这是低于0.5％。绿色荧光蛋白在单核细胞和橙色染料中性粒细胞的强度是如此明亮，通常为0.3％就足够了。注意：此表示0.15〜0.25毫瓦与我们的系统的激光功率。 </li><li>收购的512×512像素（ 即 635微米）的2.2秒的图像，使用倒置显微镜的20X干客观开放的针孔。使用10的Z深度20微米，以确保足够的信号与低功率的激光。 注：我们通常用做实验用12至16的Z深度56，M。在呈现附图和电影的情况下，在z深度为20微米。相衬允许内皮墙壁的识别和成像肠系膜上静脉进行。由于巡逻细胞移动相当缓慢，5-10微米/分钟6的速度，记录的关注的一个领域，每20秒令人满意。所描述的设置，电动阶段允许感兴趣多达7字段的同时记录。 </li><li>鼠标放置在显微镜的恒温室中的肠系膜血管被允许他们从获取的第一部电影的前准备30分钟恢复。在此期间，选择感兴趣的几个领域。重点放在最接近目标容器的腔表面。如果需要，采取轻微的自体荧光血管内皮细胞的优势，设置焦点。 注：鼠标准备在手术过程中稍微强调内皮。因此，轧制中性粒细胞和/或单核细胞所用的第一15分钟以下鼠标制剂中观察到。 </li><li>录制的第一部电影稳态条件下30分钟，一张图片，每20秒。 注：该软件获取的每个感兴趣字段在2.2秒和电动阶段自动地从一个场移动到另一个。这又回到了在20秒内的第一个位置，在记录电影的30分钟。 </li><li>要启动血管炎症，加入20微升PBS含100微克TLR2的下降/ TLR1激动剂Pam3CSK4 12直接到成像的船只。 </li><li>控制聚焦为每个感兴趣的领域。 注：在收购的过程中，总有重点的对Z轴的细微变化。尽管10-20微米的z深度，这会导致降低的荧光强度的。因此，手动重新集中的每个感兴趣字段在每部电影的最后，如果需要的话。 30分钟时段和最多3个小时，拍摄电影TAL炎症开始后。 </li><li>在实验结束时，通过颈脱位牺牲麻醉小鼠。 </li></ol> 6.代电影文件和白细胞跟踪注：尼康采集软件生成.nd2文件，但下面的程序将与其他软件和品牌的同类。 <ol><li>文件导出为TIFF系列。 </li><li>转到ImageJ的软件<a href="http://rsb.info.nih.gov/ij/">（http://rsb.info.nih.gov/ij/）。</a>导入TIFF系列。每个信道为不同的文件。 </li><li>具有2.0像素为绿色和红色通道，以减少背景噪声（处理过程&gt;滤波器&gt;中位...）的半径应用中值滤波器。 </li><li>文件转换成二进制（流程&gt;二进制&gt;进行二进制）。启用软件来计算阈值的每个图像。 </li><li>合并频道合并为一个图像序列（图像&gt;彩色&gt;合并通道...）。选择格力的单核细胞n个信道，在红色通道，并在灰色通道的透射光的中性粒细胞。 </li><li>堆叠图像转换为RGB颜色（图像&gt;颜色&gt;堆栈RGB）。 </li><li>文件保存为.AVI </li><li>数据被导入并在了Imaris软件（位平面）编制。单核细胞和中性粒细胞的运动即期跟踪向导，然后跟踪。 </li></ol>

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The authors declare that they have no competing financial interests.

在这个手稿中描述的方法提供了一个一致的方法来有效地研究单核细胞和中性粒细胞行为的稳态和炎症的条件下肠系膜静脉。 制剂的关键步骤是用PBS-湿的薄纸肠的固定。如果执行得当，肠系膜血管是很好的暴露在盖玻片进行图像采集。这使得感兴趣的几个领域的选择，监测肠系膜静脉白细胞行为。 我们的方案涉及肠系膜血管的外化的实验开始前，使（1?...

<html>嗜中性粒细胞和单核细胞是先天免疫系统，它在血液中循环连续的细胞。在损伤或感染，炎症信号诱导白细胞血细胞渗出到受损和感染的组织中，本文引发细胞免疫应答1-3。白细胞动员的快速性决定了免疫应答的积极成果。这些复杂的过程依赖于特定的分子 （如选择素，血管内皮细胞结合的趋化因子）出现在发炎的内皮细胞，帮助循环白细胞和血管内皮细胞1-3之间建立接触胶。要得到牵连白细胞分子见解招募级联，可视化细胞募集的动力学和跟踪每个小区/群体的行为是重要的。一种有效的方法包括在表达荧光蛋白在感兴趣的细胞的转基因小鼠活体显微镜。 内容“&gt;迄今为止，使用活体显微镜几种方法被开发以图像脉管4,5，例如，耳真皮脉管或肠系膜静脉由活体共聚焦显微镜成像被用来确定的鼠的Ly6C 低的单核细胞和人巡逻行为CD14 变暗 CD16 +单核细胞上的血管的稳定状态条件下6-8腔侧，鼠标提睾脉管模型通常用于监测炎症或缺血性病症中的转基因小鼠嗜中性粒细胞之下或炎症的Ly6C 高的单核细胞的行为。在这情况下，提睾通过阴囊内注射IL1β，CCL2，TNFβ或fMLP的刺激后2-4小时，组织，然后手术形象化和活体共聚焦显微镜9-11分析。 以下方案描述了一种方法来监控单核细胞和中性粒细胞的同时与任何倒置荧光共聚焦显微镜。此外，我们的方法，详细介绍了如何前映像相同的容器（稳态条件）和炎症后如何遵循白细胞募集的动力学。为此目的，我们使用在CX 3 CR1 GFP /重量鼠标，其单核细胞表达eGFP的，静脉注射一种橙色色素标记的鼠中性粒细胞。使用倒置共聚焦显微镜，它是可能的（1）来跟踪和分析稳态条件和（2）以符合两个单核细胞和中性粒细胞在同一容器中的后局部炎症招募下巡逻的Ly6C 低的单核细胞。在这里，我们创建了炎症的使用TLR2 / TLR1激动剂Pam3CSK4 12。而且，这样的成像可有助于确定是否对特定敲除小鼠或在阻断抗体6,9,13的存在下进行的，在各个步骤的白细胞募集级联感兴趣的特定分子的作用。

<table>
	<tbody>
		<tr>
			<td>5 mL polystyrene round bottom tubes&#160;</td>
			<td>Beckton Dickinson</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>10x CFI Plan Apochromat 0.45&#160;&#160; DT:4mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20x CFI Plan Apochromat VC 0.75&#160;&#160; DT:1mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Tracker Orange CMRA Dye</td>
			<td>Life Technologies</td>
			<td>C34551</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>Stem Cell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse Neutrophil Enrichement kit</td>
			<td>Stem Cell Technologies</td>
			<td>19762</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>PAA</td>
			<td>A15-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil Type A</td>
			<td>Nikon</td>
			<td></td>
			<td>any viscous oil&#160;</td>
		</tr>
		<tr>
			<td>Life Box Temperature Control System</td>
			<td>Life Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon</td>
			<td>A1R</td>
			<td>inverted microscope, motorized x/y/z stage, NIS elements software</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>phenol red free DMEM/F12</td>
			<td>Life Technologies</td>
			<td>21041-025</td>
			<td>any phenol red free medium is suitable</td>
		</tr>
		<tr>
			<td>PAM3CSK4</td>
			<td>Invivogen</td>
			<td>tlrl-pms</td>
			<td>reconstitute in PBS</td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>Stem Cell Technologies</td>
			<td></td>
			<td>included in EasySep Mouse Neutrophil Enrichement kit</td>
		</tr>
		<tr>
			<td>tissue culture dish 100</td>
			<td>TPP</td>
			<td>93100</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging neutrophils and monocytes in mesenteric veins by intravital microscopy on anaesthetized mice in real time

有效的免疫反应是依赖于血液中的白细胞的快速动员到感染或损伤的部位。 调查白细胞迁移体内对于理解白细胞跨内皮迁移和相互作用与血管内皮的分子基础是至关重要的。一种有效的方法包括在表达荧光蛋白在感兴趣的细胞转基因小鼠活体显微镜。 在这里，我们提出了一个协议，用于成像的单核细胞和中性粒细胞在CX 3 CR1 GFP /鼠标重量静脉注射橙色染料标记的嗜中性粒细胞用倒置的共聚焦显微镜。时间推移电影收集从30分钟到几个小时的成像允许的白细胞行为在肠系膜静脉两个稳态和炎性条件下分析。我们还描述的步骤，以在本地引起血管炎症TLR2 / TLR1激动剂Pam3SK4和监控subsequeNT招聘中性粒细胞和单核细胞。 所提出的技术也可以用于监测白细胞的其他人口和调查使用等刺激或转基因小鼠的分子参与了白细胞招聘或贩运。

手稿描述了一种优化的协议来方便地监视单核细胞和嗜中性粒细胞中的实时麻醉的小鼠的肠系膜静脉的行为。使用37℃-恒温室的是强制性的，以保持鼠标的温度，也由于白细胞的温度相关的运动。鼠标的制备显示于图1中。图2示出了所有在显微镜下看到的面积。透射光使肠系膜静脉（红色箭头）和动脉（蓝色箭头）的标识。 治疗ImageJ的软件生成的视?...

Watch this Scientific Journal Video about Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time at JoVE.com

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

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 ...

imaging-neutrophils-monocytes-mesenteric-veins-intravital-microscopy

Research

JoVE Journal

Immunology and Infection

10.4K 次观看.  University of Geneva. 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.

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

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 ...

科研

免疫与感染

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

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 ...

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

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

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

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

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

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

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, ...

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 ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. 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. ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

通过体内显微镜评估的小鼠中白细胞肌渗透

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...