我们特别感谢S. Graewe和五Heussler的初始培训和疟疾寄生虫的活体显微镜连续输入，J ·伯恩斯捐赠GFP转基因寄生虫，A.博世（共焦单位，CCIT - UB，IDIBAPS）在图像分析和定量和技术援助，以体育Astola的援助。我们感谢R. Tous的和一视频制作Caralt。 MF是从加泰罗尼亚的一般性的研究生奖学金的收件人。羟基磷灰石是ICREA研究教授。在HAP的实验室的工作是由欧洲共同体的第七框架计划（FP7/2007-2013）根据赠款协议ñ ° 242095的私人基金会左旋（加泰罗尼亚，西班牙），由西班牙科学和创新部（ SAF2009 - 07760）。

这种方法是在7日报道，研究使用。 1。动物与绿色荧光蛋白（GFP）转基因寄生虫感染<ol><li> P.约氏疟原虫 - GFP转基因17XL和17X线使用相同的载体，定位策略和协议描述为 P 伯氏 8。他们表示下普遍存在的P.推动者9 GFP的突变3变 ​​种疟原虫延伸因子1（Pbeef1a），指示组成型表达GFP的寄生虫在红细胞内发育周期的整个细胞质中。 </li><li>注入动物腹腔内寄生的红细胞P.（pRBCs）约氏疟原虫 - GFP转基因株系17XL和17X，从供体小鼠的尾巴的血液在5-10％原虫和PBS稀释。 5X10 5 pRBCs /鼠标的使用剂量达到1％的外围原虫后3天，infecti（PI）。 </li><li> PI在第3天，检查与两个寄生虫线感染小鼠的原虫是相同的，通过做血液涂片用姬姆萨染色和与100X的油客观光显微镜下观察的尾巴血液下降。原虫估计约300红细胞光学领域中的红细胞总数的pRBCs比例计算。 </li><li>可用于注射用FITC标记的红细胞的控制动物的特点，在正常脾脏，这些细胞的运动。 </li></ol> 2。 FITC和注射，以控制动物的红血细胞的标记<ol><li>通过一个BALB / c小鼠心脏穿刺收集200μL的1毫升血总用含乙二胺四乙酸（EDTA）（100克/升，pH值7.4），并在PBS / EDTA洗涤红细胞颗粒（0.1克/ L，pH 7.4）中，通过离心5分钟，在室温（RT）在300 XG（分）。 </li><li>在300和200μL红细胞沉淀重悬万亩; PBS / EDTA（0.1克/升，pH值8）含有FITC标记（10克/升），并在黑暗轻轻摇动2小时在室温下孵育升。此后，上清液被删除，细胞洗五次PBS / EDTA（0.1克/升，pH值7.4）（300 XG，5分钟，RT）。 </li><li>在体内实验中，稀释10μLFITC标记的红细胞沉淀在200μL的PBS，注入静脉注射到BALB / c小鼠，以达到1％的FITC标记的红细胞在循环。 </li></ol> 3。外科手术<ol><li>准备注射麻醉剂100毫克/公斤的氯胺酮和5毫克/公斤的蓝精灵每剂量根据动物的重量组成。小鼠腹腔注射一剂麻醉剂。 Readminister一半的剂量每30分钟保持鼠标全职麻醉。 </li><li>保持鼠标的温暖和验证，鼠标是完全麻醉按捏，然后再继续垫脚（通常是5-20分钟后）。 </li><li>为了在实验的过程中促进静脉给药的物质，cannulate尾静脉鼠标使用27G套管。检查，以及静脉内注射生理盐水缓冲液20-50定位针，并用胶带密封。如果它阻碍，重复静脉插管上游。注意不要引入气泡。 </li><li>通过在动物的左背侧皮肤和肌肉的小切口，暴露脾下的一部分。将少呼吸运动是观察脾，适用于暴露的表面，以保持它的清洁鼠标的头发和水合PBS。 </li><li> 60x24mm的掩护滑氰基丙烯酸酯胶粘剂（超级乐泰胶3）脾周围皮肤，让可视化密封。 </li></ol> 4。成像在脾脏的生活寄生虫<ol><li>显微镜活体实验进行了Leica TCS - SP5的共聚焦显微镜（Leica微，德国海德堡）配备孵化温度控制系统，APO 63X甘油浸泡物镜（NA 1.3），在8000线/ s和氩气（488纳米）和氦氖（594纳米，633纳米谐振扫描仪）激光器。可能需要额外的激光器，如蓝色二极管（405纳米）和二极管泵浦固态（561纳米），表1中列出的探针的激发。 </li><li>盖下滑脾朝下的目标，放在显微镜阶段的动物。脾脏微循环障碍结构的一个普遍的看法，可以选择可视化使用20倍的目标。红细胞反射对比将有助于选择不同地区的利益，在更高的放大倍率之后形象。 </li><li> 63X甘油浸泡的目的，采用组织自体荧光的镜头聚焦感兴趣的选定区域。 GFP寄生虫观察经过不同领域的脾。 </li><li>荧光是记录在两个不同的茶nnels（激发/发射波长488/505-580 FITC / GFP和488/570-630组织自体荧光纳米的纳米）与针孔设置为3.0艾里单位。红细胞反射（488/480-495纳米），连同荧光染料标记的血液血管（见表1），用于成像和血流下面描述的实验区获得更多的信息。 </li><li>通过五个捕捉图像的Z堆栈覆盖，因为立体感的器官8微米的深度，速度为8 kHz，1.5分钟生成的视频。 </li><li>脾进行定量分析不同区域的记录影片。 </li></ol> 5。健脾和微血管血流量测量的图像采集活体显微镜<ol><li>溶解在生理盐水中的重要的荧光探针，可以到尾静脉注射在血管和洞察到脾的结构图像的实验。列表A探针及其应用是在表1 10。 </li><li>标签用荧光葡聚糖的血管系统，准备70 kDa的葡聚糖与德克萨斯红标记在100μL缓冲液1毫克。 </li><li>使用空心尾静脉注入荧光葡聚糖成像动物。 </li><li>横向设置的船只，在激光扫描的方向，光场的旋转（不影响速度）。使用XY和XT线在该船只的中央管腔扫描模式。使用双向扫描速度为8 kHz，获得一个512 × 512像素的图像线平均为32。 </li><li>船只在三个不同的渠道（激发/发射波长488/505-580纳米，594/605-660纳米，FITC / GFP，右旋糖酐德克萨斯红和红细胞反射，分别488/480-495纳米）获取图像。 </li><li>以不同直径和心动周期不同阶段的船只图像，以弥补波动。在这些图像，移动细胞产生的条纹将被用来量化血流11。 </li></ol> 6。图像处理和寄生虫的流动性的定量分析，使用ImageJ软件<ol><li>创建一个使用ImageJ软件（版本1.39o，韦恩Rasband，国立卫生研究院，生成图像序列实时视频<a href="http://www.macbiophotonics.ca">www.macbiophotonics.ca</a> ）。 </li><li>打开“LIF”ImageJ中的文件保持“xyzct”序列和分离通道。 </li><li>注册从元数据文件中的一些有用的信息：dblvoxelX体素宽，dblvoxelY体素高，dblvoxelZ体素的深入和帧之间连续的Z -帧和协议栈之间的间隔。这些信息将被用于校准。 </li><li> GFP寄生虫图像（通道1）减去自体荧光（通道2）。筛选与高斯模糊= 1图像。请记得，在图像中使用高斯模糊滤镜需要PUBLICATIO宣布NS。保存文件“animal1_m1_substract.seq”。 </li><li> Z -编码的彩色视频创建为支持寄生虫流动性的定量分析材料，因为这将促进在快速移动的粒子和Z轴的运动特性的单粒子鉴别。 </li><li>堆栈转换图像5D，第三维Z和第四维时间。每个Z和覆盖不同的颜色。 </li><li>项目所有的Z使用所有的时间帧的最大强度，以创建一个Z -编码的彩色视频。另存为“animal1_m1_Z_color.avi”。 </li><li>分类和标签出现在视频的第10帧的时间，根据居住的帧数量（从1到10）的所有粒子。在每部影片中，20粒子将跟踪以下所得的比例。共120寄生虫3只动物将被量化，使用6个视频/动物代表不同区域的脾。 </li><li>报告对每个Z居住帧在整部影片是可以量化的所有粒子。 </li><li>执行4D（X，Y，Z，T）手册使用MTrackJ插件（大肠杆菌Meijering书面）的粒子跟踪。打开文件图片5D“animal1_m1_substract.seq”，并设置图像的属性，使用像素宽度，高度，深度（微米）和堆栈的时间间隔（秒）之前注册的信息。配置轨道设置如下：“移动到下一次”和“适用于本地光标明亮centroid/25x25pixel”。配置显示：“起源”，“形象展示”，“显示积极跟踪”，“只显示电流通道的轨道”，“只显示在当前时间点跟踪”。 </li><li>添加为每个粒子的轨道。考虑排量只有高于6微米（平均直径为PRBC）在Z轴的运动。按照超过100帧的最大的粒子。 </li><li>保存的X，Y，Z和“animal1_m1_p1”从轨道测量吨的坐标。XLS。 </li><li>位移的措施（D = SQRT（（X决赛- X 头文字 ）2 +（Ÿ 最后 - Y 头文字 ）+（Z 最后 - Z 头文字 ）2）;路径长度（P =ΣN = 0→最终 SQRT（（X N +1 - X N）2 +（ 1 - Y Y N N）2 +（Z N +1 Z N）2）用n表示每个位置跟踪;平均流速和停留时间可使用X，Y，值计算Z，吨坐标跟踪校准根据登记的数据。粒子的方向性定义为位移与路径长度的智商值接近1表示定向运动和价值观接近0表示克制运动12一个计算模板是推动封闭。 </li></ol> 7。体积血流量的计算<ol><li>体积血流量Q = V *π*深V 2 / 4，V字形，红细胞流速在横截面一个估计ND D V，管腔血管直径11。 </li><li>要计算V，测量角度（θ）五粒子呈现出明亮的反射（RBC）和4个粒子的条纹，在每个XT使用ImageJ软件的图像显示绿色荧光（PRBC - GFP ）的条纹。 XY图像的流明测量血管直径。 </li><li>则表示速度为V = 1/tan（θ）* D E / D V至正常化红细胞（D E = 6微米）和管腔的血管直径。 </li><li>量化至少三个不同直径的船只，每艘船五XT图像。 </li></ol> 8。统计分析<ol><li>进行统计分析，积的方向性，平均密度分布的速度和停留时间和使用STATA（IC10）在平等的中位数测试，以评估两者之间的寄生虫线差异。 </li></ol> 9。代表性的成果活体IMA蔓茎在脾GFP的寄生虫透露流动性寄生虫两个菌株之间的差异。单寄生虫的流动性参数的定量分析表明，降低速度，缺乏方向性和增强17X株感染小鼠的寄生虫的停留时间。此外，船只体积血流量没有改变菌株间7。图1A的技术程序。图1B显示了一个用FITC标记的红细胞注入鼠标的正常脾的总的看来，在进入红髓变焦和其他的船只（图1B，放大，分别在1和2）。血管被证明由70 kDa的葡聚糖得克萨斯红一起注射红细胞反射对比。在表1中总结的其他荧光染料，可用于获取信息的器官被成像，如赫斯特（图1C，1D），。 17XL和17X应变寄生虫实时成像是在1和2电影，一些17X - PRBC（电影2）呈现出滚环的行为。流动性参数的定量分析是通过跟踪个人寄生虫的Z -编码的彩色图像的帮助。图2A显示了一个Z -编码的颜色栈，包围粒子出现在不同的平面移动Z轴投影。图2B和2C的曲目，分别代表不同的17X和17XL感染寄生虫。的图2D和2E寄生虫人口密度分布图，分别从方向性和停留时间的所有量化颗粒结果。为了监视在使用活体显微镜脾血流量，从红细胞运动船只的中央管腔XT图像获得的条纹进行测定，计算速度11。图像显示XY相应的XT扫描线（图2G）的船只的扫描（图2F）。 <table border="1"><tbody><tr><td> 荧光PROBË </td><td> 本地化 </td><td> 1光子激发（NM） </td><td> 2光子激发（NM） </td><td> 检测排放（NM） </td><td> 数量/鼠标重量 </td></tr><tr><td>赫希斯特33342 </td><td>膜permeant DNA结合探头。标签静脉注射后的所有核细胞（活的和死的）。 </td><td> 405 </td><td> 800 </td><td> 410-480 </td><td> 12.5克/公斤</td></tr><tr><td>碘化丙啶</td><td>膜impermeant DNA结合探头。它的标签损害膜细胞凋亡和坏死细胞的细胞核。 </td><td> 561 </td><td> 800 </td><td> 570-650 </td><td> 250毫克/千克</td></tr><tr><td> 70000 MOL WT葡聚糖荧光（FITC，德克萨斯州红） </td><td>流体相的标志物，提高血浆的对比。 </td><td> FITC标记的488得克萨斯州红594 </td><td> 800 </td><td> 500-540 600-650 </td><td> 50毫克/千克</td></tr><tr><td>荧光素钠</td><td>散装流体相白蛋白标记，提高血浆的对比。 </td><td> 488 </td><td> 800 </td><td> 500-540 </td><td> 2毫摩尔/千克</td></tr><tr><td>伊文思蓝</td><td>散装流体相白蛋白标记，提高血浆的对比。 </td><td> 633 </td><td> ND </td><td> 645-700 </td><td> 20毫克/千克</td></tr><tr><td>若丹明R6 </td><td>活跃​​的线粒体中积累的重要探头。它的标签后，静脉注射白细胞内皮细胞和循环。 </td><td> 561 </td><td> 800 </td><td> 570-650 </td><td> 25毫克/千克</td></tr><tr><td> Fluospheres - 1微米直径</td><td>珠是由细胞吞噬活性摄取。 </td><td> 488 </td><td> 800 </td><td> 500-540 </td><td> ND </td></tr><tr><td> Alexa488标记的纤维蛋白IIβ链特异性抗体</td><td>探测器标签纤维素IIβ链</td><td> 488 </td><td> 800 </td><td> 500-540 </td><td> 0.3毫克/千克</td></tr></tbody></table> 表1：活体显微镜荧光探针。标签脾在体内，可用于不同的本地化至关重要的荧光染料。激发/发射（EXC / EM）的范围与单光子（或双光子显微镜）提供使用。剂量表示溶解在缓冲液0.1-0.2毫升和鼠标的尾静脉注射。 [ND：在这项研究中确定。 <img src="/files/ftp_upload/3609/3609fig1.jpg" alt="图1" /> 图1。脾的活体显微镜。答：徕卡TCS - SP5共聚焦显微镜显微镜舞台上放置一个鼠标。鼠标B.形象代表面积的非感染动物注射用FITC标记的红细胞脾脾下部封面滑揭露和密封。70 kDa的葡聚糖德克萨斯红的血管可视化。反射（黄色），葡聚糖（蓝色）和FITC标记的红细胞（绿色）所示。在打击白框代表（1）开放式循环和密切循环（2）地区。开放式循环（C）和关闭循环（四）与70 kDa的葡聚糖（红色）和Hoechst 33342（蓝色）染色。 <img src="/files/ftp_upload/3609/3609fig2.jpg" alt="图2" /> 图2。寄生虫的流动性和血流量。 交流的量化。粒子的运动定量分析的四个维度（4D）便利通过使用颜色编码的图像处理。A.跟踪的Z -编码的颜色代表使用5个不同深度的最大强度投影图像，深入的信息进行。白色矩形代表一个时间点的不同Z相同的粒子。不同的位置，由于采集时间之间的失误不同Z图像。深度代码：黄色（1微米），橙（2微米），粉红（4微米），蓝（6微米），绿（8微米）B，C.粒子运动的时间推算，每个时间间隔颜色：灰色（0-2.4秒），青色（2.4-4.8秒），洋红（4.8-7.0秒），红（7.0-9.4秒）和黄色（9.4-11.8秒）。白线代表4D手动跟踪使用MTrackJ D，E GFP的颗粒密度分布的方向性值（四），17X（11.8）（B）和17XL（4.8秒）（三 ）绿色荧光蛋白寄生虫颗粒停留时间（E）。数据对应的每一行从与平等中位数测试分析的三个独立实验的寄生虫和100 FITC标记的红细胞120颗粒。 17X/17XL/FITC-RBCs位数0.53/0.75/0.85（D）和两线之间的差异4.61/0.67/0.9秒（五）。 <st<html>荣&gt;（D）和（五）有统计学显着性 （P &lt;0.001 ）。 FITC标记的红细胞和17XL寄生虫之间的差异没有统计学意义（P&gt; 0.05），F，G.脾虚血流量测量。从同一艘船（白线）的中央管腔行扫描XY图像（F）和XT图像（G）的代表。脾血管与70 kDa的葡聚糖（红色），PRBC（绿色）和红细胞的反思（蓝色）血浆。 （2）电影1和2。定时17XL（1）或17X感染小鼠脾活体显微镜图像GFP转基因在10％原虫寄生虫（Z轴的最大投影）。寄生虫和组织自体荧光显示在绿色和红色分别。标尺代表10μm和秒的时间间隔。 V“&gt;点击这里观看电影1。 <a href="/files/ftp_upload/3609/3609movie2.mov">点击这里观看电影2。</a>

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Commun. 2, 165-165 (2011).</li><li>Martin-Jaular, L., Ferrer, M., Calvo, M., Rosanas-Urgell, A., Kalko, S., Graewe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-specific+spleen+remodelling+in+Plasmodium+yoelii+infections+in+Balb/c+mice+facilitates+adherence+and+spleen+macrophage-clearance+escape.">Strain-specific spleen remodelling in Plasmodium yoelii infections in Balb/c mice facilitates adherence and spleen macrophage-clearance escape.</a> Cell. Microbiol. 13, 109-122 (2011).</li><li>Linden, M. v. a. n. d. e. r., R, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Plasmodium+berghei+reference+line+that+constitutively+expresses+GFP+at+a+high+level+throughout+the+complete+life+cycle.">A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle.</a> Mol. Biochem. Parasitol. 137, 23-33 (2004).</li><li>Cormack, B. P., Valdivia, R. H., Falkow, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS-optimized+mutants+of+the+green+fluorescent+protein.">FACS-optimized mutants of the green fluorescent protein.</a> 173, 33-38 (1996).</li><li>Dunn, K. W., Sandoval, R. M., Kelly, K. J., Dagher, P. C., Tanner, G. A., Atkinson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+studies+of+the+kidney+of+living+animals+using+multicolor+two-photon+microscopy.">Functional studies of the kidney of living animals using multicolor two-photon microscopy.</a> Am. J. Physiol. Cell. Physiol. 283, C905-C916 (2002).</li><li>Zhong, Z., Petrig, B. L., Qi, X., Burns, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+erythrocyte+velocity+and+retinal+blood+flow+using+adaptive+optics+scanning+laser+ophthalmoscopy.">In vivo measurement of erythrocyte velocity and retinal blood flow using adaptive optics scanning laser ophthalmoscopy.</a> Opt. Express. 16, 12746-12756 (2008).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Bowdler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The complete spleen. , (2002).</li><li>Grayson, M. H., Hotchkiss, R. S., Karl, I. E., Holtzman, M. J., Chaplin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+comparing+T+lymphocyte+trafficking+to+the+spleen+and+the+mesenteric+lymph+node.">Intravital microscopy comparing T lymphocyte trafficking to the spleen and the mesenteric lymph node.</a> Am. J. Physiol. Heart. Circ. Physiol. 284, H2213-H2226 (2003).</li><li>Khandoga, A. G., Khandoga, A., Reichel, C. A., Bihari, P., Rehberg, M., Krombach, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+and+quantitative+analysis+of+leukocyte+directional+migration+and+polarization+in+inflamed+tissue.">In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue.</a> PLoS. One. 4, 4693-4693 (2009).</li><li>Weiss, L., Geduldig, U., Weidanz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+splenic+control+of+murine+malaria:+reticular+cell+activation+and+the+development+of+a+blood-spleen+barrier.">Mechanisms of splenic control of murine malaria: reticular cell activation and the development of a blood-spleen barrier.</a> Am. J. Anat. 176, 251-285 (1986).</li><li>Swirski, F. K., Nahrendorf, M., Etzrodt, M., Wildgruber, M., Cortez-Retamozo, V., Panizzi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+splenic+reservoir+monocytes+and+their+deployment+to+inflammatory+sites.">Identification of splenic reservoir monocytes and their deployment to inflammatory sites.</a> Science. 325, 612-616 (2009).</li><li>Grayson, M. H., Chaplin, D. D., Karl, I. E., Hotchkiss, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+fluorescent+intravital+microscopy+of+the+murine+spleen.">Confocal fluorescent intravital microscopy of the murine spleen.</a> J. Immunol. Methods. 256, 55-63 (2001).</li><li>Bajenoff, M., Glaichenhaus, N., Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblastic+reticular+cells+guide+T+lymphocyte+entry+into+and+migration+within+the+splenic+T+cell+zone.">Fibroblastic reticular cells guide T lymphocyte entry into and migration within the splenic T cell zone.</a> J. Immunol. 181, 3947-3954 (2008).</li></ol>

没有利益冲突的声明。

脾在这种啮齿动物疟疾模型的活体显微镜的实施开通过这个到现在为止一直被认为是“黑盒子”由于技术方面的考虑机关调查寄生虫的动态通过的可能性。在这里，一个重要的努力，以适应定量的方法，它允许在单一和人口的水平比较分析，不同的寄生虫线。相反，其他组织和细胞已疟疾3,5前成像，成像通过pRBCs通过脾需要考虑到三个维和条块分割的器官，不同的环流存在，快与慢13</...

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intravital microscopy of the spleen: quantitative analysis of parasite mobility and blood flow

在实验啮齿动物疟疾模型活体显微镜的问世，使寄生虫-宿主相互作用 1,2的知识的重大进展。因此，在预红细胞阶段疟原虫体内成像显示到皮肤淋巴结3，寄生在皮肤4的完整的开发，并形成，以保证移民的一个肝细胞衍生merosome寄生虫的积极入口裂殖子释放进入血液流5。此外，个人寄生虫在红细胞的发展，已最近记录使用4D成像和挑战我们目前蛋白在疟疾6出口。因此，活体成像，从根本上改变我们对疟原虫发展中的关键事件。不幸的是，疟疾寄生虫通过脾，一个主要的淋巴器官的动态通过的研究精美的适应，以清除感染的红Blood细胞缺乏由于技术上的限制。 BALB / C小鼠约氏疟原虫疟疾的小鼠模型，我们已经实施了脾的活体成像和屏障红髓中的成纤维细胞的起源细胞，期间报告的差重塑和坚持寄生的红细胞（pRBCs）与非致命性寄生虫线P.yoelii 17X感染，而不是P.yoelii 17XL致命寄生虫线7感染。为了达到这些结论，一个特定的方法，使用ImageJ免费软件开发，使快速立体单pRBCs运动的特性。获得的结果与本议定书允许速度，方向和居住的寄生虫的时间确定在脾，所有参数，解决在体内坚持。此外，我们报告的血流量，用活体显微镜量化和不同的使用方法着色剂，以获得洞察到复杂的微循环结构的脾ferent。 伦理声明 在巴塞罗那大学动物设施在巴塞罗那CEEA瑞银（协议没有DMAH：5429）大学动物实验伦理委员会批准的准则和协议的规定，所有的动物实验研究。从查尔斯河实验室获得6-8周龄雌性BALB / c小鼠。

Watch this Scientific Journal Video about Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow at JoVE.com

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

我们使用GFP转基因疟疾寄生虫和寄生虫的流动性和血流量在本机关的量化脾的活体显微镜的方法。

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-spleen-quantitative-analysis-parasite-mobility

Research

JoVE Journal

Immunology and Infection

18.1K 次观看.  Barcelona Centre for International Health Research. 我们使用GFP转基因疟疾寄生虫和寄生虫的流动性和血流量在本机关的量化脾的活体显微镜的方法。

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

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

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 ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

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 ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...