Name: use of an optical trap for study of host-pathogen interactions for dynamic live cell imaging
Uploaded: 2011-07-28T12:00:00
Description: Watch this Scientific Journal Video about Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging at JoVE.com

这项工作得到了马萨诸塞州总医院医学内部资金部（JMT，MKM的MLC，JMV），国家生物医学成像和生物工程授予T32EB006348（CEC），美国马萨诸塞州总医院的中心计算和综合生物学的发展基金和AI062773（ RJH），补助AI062773，DK83756，和DK 043351（RJX），0643745（MJL）美国国家科学基金会，美国国立卫生研究院R21CA133576（MJL），以及国家过敏和传染病研究所（NIAID的）美国国立卫生研究院（NIH）的AI057999（JMV ）。我们感谢尼古拉三尤德，有益的讨论和查尔斯毛毡（RPI的公司）提供技术援助。

1。光学诱捕病原体的文化条件<ol><li>生长在一个半固体琼脂的SBD（沙氏葡萄糖）媒体30 ° C 曲霉 （B-5233/RGD12-8）3天。 </li><li>成长C。白色念珠菌 （SC5314）在YPD（酵母蛋白胨葡萄糖）液体培养基在摇床培养箱含有100μg/ mL氨苄青霉素一夜之间30 ° C。 </li></ol> 2。病原体为荧光标记的制备<ol><li>收获所需量的病原体，并转移到1.5 ml反应管。 </li><li>反应管中加入300μL磷?...

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在这项工作中，我们使用了一个光学陷阱捕捉到3微米之间的尺寸的病原体 - 5微米。虽然我们的实验室感兴趣的病原体通常有这些方面，这里所描述的光镊系统是灵活的陷阱大范围的大小。事实上，光学陷阱被用来捕捉范围从单个原子，细胞直径约10微米的颗粒。此外，这种光学诱捕系统能够捕捉到各种形状：球形的颗粒，椭圆形，而且非常细长的颗粒，这是有用的生物病原体时。 <p class="jove_c...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> 曲霉 </td><td></td><td></td><td>白化菌株，B-5233/RGD12-8，KJ权涌，美国国立卫生研究院的礼物</td></tr><tr><td>白色念珠菌</td><td></td><td></td><td> SSY50 - B埃莱夫塞里奥Mylonakis，麻省总医院的礼物突变体; SC5314应变，从杰拉尔德芬克的礼物，怀特黑德研究所</td></tr><tr><td>的Alexa Fluor 488 </td><td> Invitrogen公司</td><td> A20000 </td><td></td></tr><tr><td>的Alexa Fluor 647 </td><td> Invitrogen公司</td><td> A20006 </td><td></td></tr><tr><td>二甲基甲酰胺</td><td>西格玛</td><td> D4551 </td><td></td></tr><tr><td>新鲜血液</td><td></td><td></td><td> RJW荒地，麻省总医院，HMS的礼物</td></tr><tr><td>尼康倒置显微镜</td><td>尼康</td><td></td><td>钛 - E型</td></tr><tr><td>补漏激光，ChromaLase </td><td>蓝天研究</td><td> CLAS - 106 - STF02 - 02 </td><td></td></tr><tr><td>荧光激发激光</td><td>连贯</td><td></td><td>模型伊诺70C </td></tr><tr><td>为诱捕组件的面包板</td><td> Thorlabs </td><td> MB1224，MB1218 </td><td></td></tr><tr><td>光学航空表</td><td>技术制造公司</td><td></td><td></td></tr><tr><td>踏板控制的电子快门</td><td> Uniblitz </td><td></td><td>购买文森特协会，纽约州罗彻斯特</td></tr><tr><td>单模光纤</td><td> OZ光学</td><td> PMJ - 3S3S - 1064 - 6 </td><td></td></tr><tr><td>光纤定位</td><td> Thorlabs </td><td> PAF - X - 5 - C </td><td></td></tr><tr><td>光纤准直器</td><td> OZ光学</td><td> HPUCO - 23 - 1064 - P - 25AC </td><td></td></tr><tr><td>为望远镜镜头</td><td> Thorlabs </td><td> AC254 - 150 - B </td><td>焦距为150毫米</td></tr><tr><td>翻译阶段（X，Y，Z） </td><td>纽波特</td><td> M - 461 - XYZ </td><td></td></tr><tr><td>红外分色镜</td><td>浓度</td><td> ET750 - SP - 2p8 </td><td></td></tr><tr><td>物镜（100X） </td><td>尼康</td><td></td><td> NA = 1.49，浸油，TIRF目标</td></tr><tr><td>共焦头</td><td>横河电机</td><td> CSU - 11 </td><td></td></tr><tr><td>偏光镜</td><td>尼康</td><td> MEN51941 </td><td></td></tr><tr><td> Wollaston棱镜</td><td>尼康</td><td> MBH76190 </td><td></td></tr><tr><td> EM - CCD摄像头</td><td>滨松</td><td> C9100 - 13 </td><td></td></tr><tr><td> CCD摄像头（ORCA的急诊室） </td><td>滨松</td><td> C4742 - 80 - 12AG </td><td></td></tr><tr><td>滤光轮</td><td> Ludl </td><td> 99A353 </td><td></td></tr><tr><td>滤光轮</td><td>萨特</td><td> LB10 -西北欧</td><td></td></tr><tr><td>墓室玻璃罩</td><td> Lab-Tek/Nunc </td><td> 155409 </td><td></td></tr><tr><td>磁珠</td><td> Invitrogen公司</td><td> 111 - 51D </td><td>涂有抗CD3 </td></tr><tr><td>贝科改良Eagle培养基（DMEM） </td><td> Invitrogen公司/ Gibco公司</td><td> 10313 </td><td></td></tr><tr><td>青霉素/链霉素</td><td> Invitrogen公司/ Gibco公司</td><td> 15140-122 </td><td></td></tr><tr><td> L -谷氨酰胺</td><td> Invitrogen公司/ Gibco公司</td><td> 25030-081 </td><td></td></tr><tr><td>胎牛血清（Hyclone公司） </td><td> ThermoScientific </td><td> SH30071.03 </td><td></td></tr></tbody></table>

use of an optical trap for study of host-pathogen interactions for dynamic live cell imaging

动态活细胞成像允许直接的免疫系统的1，2细胞之间的实时交互的可视化;然而，在空间和时间之间的吞噬细胞和微生物控制的缺乏已呈现集中观察到病原体的宿主反应的初步相互作用困难。从历史上看，如细 ​​胞的吞噬功能3间接触事件已经通过混合两种类型的细胞成像，然后连续扫描场的观点，找到适当的互动阶段偶然间接触。这些事件的随机性，使这个过程繁琐，它是通过这种方法难以观察到在细胞与细胞接触的早期或稍纵即逝的事件。这种方法需要发现细胞对接触的边缘上，并观察他们，直到他们完善他们的联系，或不。为了解决这些限制，我们使用光学诱捕作为一种非侵入性，非破坏性的，但快速和有效的方法定位在培养细胞。 在生物学研究中越来越多地利用光学陷阱，或光镊，捕捉和物理操纵细胞和其他微米大小的颗粒在三个层面 4 。首先观察到的辐射压力和应用光镊系统于1970年5，6，最初是用来控制在1987年7的生物标本。从那时起，光镊技术已经成熟到探头8-13多种生物现象。 我们描述的方法14，进步活细胞成像，通过与纺丝温度和湿度控制的磁盘共聚焦显微镜，提供精致的空间和时间在生理环境中病原微生物的控制，以方便与宿主细胞相互作用，确定由一个集成光学陷阱运营商。活，如白色念珠菌和曲霉 ，这可能会导致免疫功能低下15个人，16（ 例如艾滋病，化疗和器官移植患者）的潜在致命性，侵袭性感染，病原微生物光学被困采用非破坏性的激光强度和移动相邻的巨噬细胞，它可以吞噬病原体。高分辨率，传输光，荧光为基础的电影的建立能力，观察活细胞的吞噬功能的早期事件。为了证明在免疫学的广泛适用性，主要的T细胞也被困和操纵与抗CD3 涂层微球在体内形成的突触和突触形成的成像时间推移也获得。通过提供一个方法来施加罚款活病原体与免疫细胞的空间控制，细胞相互作用可以被捕获，以最小的扰动细胞荧光显微镜和先天免疫和适应性免疫的早期反应可以产生强大的洞察力。

Watch this Scientific Journal Video about Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging at JoVE.com

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

一种方法是描述单独选择，操作和使用光学陷阱，再加上一个旋转的磁盘显微镜图像活病原体。光陷阱提供了空间和时间的控制生物体和地方他们相邻的宿主细胞。荧光显微镜捕捉动态，以最小的扰动细胞间的相互作用。

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

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