Name: in vivo imaging of tumor angiogenesis using fluorescence confocal videomicroscopy
Uploaded: 2013-09-11T14:30:00
Description: Watch this Scientific Journal Video about In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy at JoVE.com

1。造影剂的制备<ol><li>为FITC-葡聚糖70kDa的，注入的剂量为500毫克/千克（10毫克的FITC-葡聚糖稀释在0.1ml生理盐水的小鼠重20克）。 </li><li>该代理不应该过长暴露在光线下。为了避免漂白，建议用铝箔覆盖的管。 </li></ol> 2。麻醉<ol><li>小鼠通过腹膜内注射甲苯噻嗪的1:4的混合物的麻醉（Rompun 2％，拜耳，皮托，法国）和氯胺酮（氯胺酮500，维克，卡罗，法国），分别为66毫克/公?...

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	<li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamental+concepts+of+the+angiogenic+process.">Fundamental concepts of the angiogenic process.</a> Curr Mol Med. 3 (7), 643-651 (2003).</li><li>Laemmel, E., Genet, M., Le Goualher, G., Perchant, A., Le Gargasson, J. F., Vicaut, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibered+confocal+fluorescence+microscopy+(Cell-viZio)+facilitates+extended+imaging+in+the+field+of+microcirculation.+A+comparison+with+intravital+microscopy.">Fibered confocal fluorescence microscopy (Cell-viZio) facilitates extended imaging in the field of microcirculation. A comparison with intravital microscopy.</a> J Vasc Res. 41 (5), 400-411 (2004).</li><li>Charnley, N., Donaldson, S., Price, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+angiogenesis.">Imaging angiogenesis.</a> Methods Mol Biol. 467, 25-51 (2009).</li><li>Faye, N., Fournier, L., Balvay, D., Taillieu, F., Cuenod, C., Siauve, N., Clement, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+contrast+enhanced+optical+imaging+of+capillary+leakage.">Dynamic contrast enhanced optical imaging of capillary leakage.</a> Technol Cancer Res Treat. 10 (1), 49-57 (2011).</li><li>Kurose, I., Kubes, P., Wolf, R., Anderson, D. C., Paulson, J., Miyasaka, M., Granger, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+nitric+oxide+production.+Mechanisms+of+vascular+albumin+leakage.">Inhibition of nitric oxide production. Mechanisms of vascular albumin leakage.</a> Circ Res. 73 (1), 164-171 (1993).</li><li>Faye, N. F. L., Balvay, D., Thiam, R., Orliaguet, G., Clement, O., Dewachter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+capillary+leakage+is+involved+in+the+onset+of+anaphylactic+hypotension.">Macromolecular capillary leakage is involved in the onset of anaphylactic hypotension.</a> Anesthesiology. , (2012).</li><li>Faye, N., Fournier, L., Balvay, D., Thiam, R., Orliaguet, G., Clement, O., Dewachter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Macromolecular Capillary Leakage Is Involved in the Onset of Anaphylactic Hypotension. Anesthesiology. 117 (5), 1072-1079 (2012).</li><li>Tozer, G. M., Kanthou, C., Baguley, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupting+tumour+blood+vessels.">Disrupting tumour blood vessels.</a> Nat Rev Cancer. 5 (6), 423-435 (2005).</li><li>Ntziachristos, V., Schellenberger, E. A., Ripoll, J., Yessayan, D., Graves, E., Bogdanov, A., Josephson, L., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+antitumor+treatment+by+means+of+fluorescence+molecular+tomography+with+an+annexin+V-Cy5.5+conjugate.">Visualization of antitumor treatment by means of fluorescence molecular tomography with an annexin V-Cy5.5 conjugate.</a> Proc Natl Acad Sci U S A. 101 (33), 12294-12299 (2004).</li><li>Cuccia, D. J., Bevilacqua, F., Durkin, A. J., Merritt, S., Tromberg, B. J., Gulsen, G., Yu, H., Wang, J., Nalcioglu, O. <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+quantification+of+optical+contrast+agent+dynamics+in+rat+tumors+by+use+of+diffuse+optical+spectroscopy+with+magnetic+resonance+imaging+coregistration.">In vivo quantification of optical contrast agent dynamics in rat tumors by use of diffuse optical spectroscopy with magnetic resonance imaging coregistration.</a> Appl Opt. 42 (16), 2940-2950 (2003).</li></ol>

肿瘤微循环的研究已成为了解肿瘤的生长，传播和对治疗的反应1的病理生理必不可少的。光学成像是可用于使用荧光造影剂，观察毛细血管和量化形态（功能性毛细血管密度）和功能性（指数泄漏）的参数的技术之一。 我们在这项研究中所使用的荧光显微成像既有优点也有限制。一个优点是能够选择所使用的造影剂的大小。这里，具有70-kDa的FITC-葡聚糖，通过在实验...

血管生成是一个复杂的过程1，涉及从预先存在的血管形成新的血管。在组织的微循环的病理变化，小动脉，毛细血管和小静脉的组成，有牵连的大范围的疾病，例如癌症，炎症，或糖尿病。因此，必须发展的方法来定量评估微血管结构和功能。成像使微血管研究在非或微创方式，在实时和体内 ，并在相同的动物2随时间重复测量。 目前，动态对比增强（DCE）成像3通常被用来评估组织的微循环。动态对比增强成像是如下一段时间静脉注射示踪剂的生物分布的技术。从此次收购，定量参数可以提取反映组织血管。 DCE成像已最经常使用的CT，MRI或超声波。然而，这些成像技术不允许直接观看的微血管，因为它们的分辨率，比使用特定的实验设备等，最常见的仍然是宏观的。 在本文中，我们提出使用动态对比增强光学成像，与纤维状的共聚焦电视显微镜来研究肿瘤血管在微观尺度和体内 。我们使用的大分子造影剂（FITC-葡聚糖），其只保持容器或泄漏内通过内皮屏障进入间质，根据其分子量和组织的血管内皮细胞的特性研究4。这使研究既微血管结构，通过正确划定的船只，和毛细血管的通透性，通过泄漏和积聚在间质。

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Insulin serynge 
 Myjector 1ml 
 29G</td>
 <td>Terumo Europe </td>
 <td>BS-05M2913</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorescein isothiocyanate-dextran 70 kDa </td>
 <td>Sigma-Aldrich</td>
 <td>01619HH</td>
 <td>100 mg/mL 
 diluted in saline</td>
 </tr>
 <tr>
 <td>Fibered confocal videomicroscopy</td>
 <td>Cellvizio - MaunaKea Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Calibration and Cleaning Kit for LEICAFCM1000</td>
 <td>Leica Microsystems</td>
 <td>LSU-488</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Probe ProFlexTM Z</td>
 <td>MaunaKea Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Mosaicing software</td>
 <td>MaunaKea Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Vessel detection software</td>
 <td>MaunaKea Technologies</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

in vivo imaging of tumor angiogenesis using fluorescence confocal videomicroscopy

纤维状共聚焦荧光体内成像用的光纤束采用相同的原理荧光共聚焦显微镜观察。它可以在原位元件通过光纤激发荧光，然后通过同一根光纤记录一些所发射的光子。光源是发出的激发光通过光纤束中的元素和，因为它扫描过样品，通过再现像素的图像的像素的激光。作为该扫描是非常快的，通过将其与专用的图像处理软件相结合，图象实时地与12帧/秒的频率可以得到。 我们开发出一种技术来定量表征毛细血管的形态和功能，使用共焦荧光电视显微镜设备。在我们的实验中，第一步是要记录5秒的电影中的四个象限的肿瘤可视化的毛细血管网。所有影片均采用软件处理（我mageCell，莫纳克亚技术，法国巴黎），执行船舶自动分段围绕一个选定的直径（在我们的案例中10微米）。因此，我们可以量化的“功能性毛细血管密度”，它是总血管面积和图像的总面积之比。这个参数是一个替代指标微血管密度，通常采用病理工具测量。 第二个步骤是，记录肿瘤的电影在20分钟内通过毛细血管壁进入间质的量化的大分子造影剂的泄漏。通过测量间质比，在血管信号强度的比率，一个人指数泄漏“获得，作为一个替代指标毛细血管的通透性。

通过分析所收集的数据，我们可以定量分析不同的参数，反映微循环。 我们研究了体内的结肠肿瘤使用纤维状的共聚焦荧光电视显微镜系统（Cellvizio，Maunakea技术，巴黎，法国2），喷射的高分子荧光对比剂后异硫氰酸荧光素-葡聚糖（植入BALB-C小鼠的外周血管网FITC-葡聚糖）与70 kDa的（Sigma-Aldrich公司，圣康丁法拉维耶，法国）中，用488 nm和520 nm处分别激发和发?...

Watch this Scientific Journal Video about In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy at JoVE.com

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

在本文中，我们提出了一个使用动态对比增强荧光电视显微镜来分析肿瘤微血管体内的方法。购入两个量化参数：功能性毛细血管密度反映了肿瘤的血管，和索引泄漏反映内皮壁的泄漏。

在肿瘤血管生成中使用荧光共聚焦电视显微镜活体成像

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