We thank Joshua Marcus for technical support and Jolien Tyler, Ph.D., Director of the Richard J. McIntosh Light Microscopy Core Facility, for technical advice. This work was supported by funds from the University of Colorado Boulder and the University of Colorado Boulder Graduate School to J.D.O. R.T.B. is partially supported by pre-doctoral training grant from the NIH (T32 GM008759). We thank Karyopharm Therapeutics, Inc. for selinexor and Merck Serono for Kinesin-5 inhibitor. FUCCI plasmids are from Atsushi Miyawaki (RIKEN, Japan) via MTA. mCherry-BP1-2 was from Addgene. HeLa expressing H2b-mCherry and &#946;-tubulin-EGFP are from Daniel Gerlich (IMBA, Austrian Academy of Sciences, Austria).

以下方案使用由实验在图4 和6关于采集设置和实验条件定义的参数。许多这些参数可以被修改以适应其它实验（ 即，曝光时间，分级，荧光通道等 ）。所有的程序必须坚持体制准则和条例，并通过生物安全委员会的批准。显微镜制造商的网站含有活细胞成像优秀的信息。 1.显微镜和图像处理软件<ol><li>在各种各样的倒置显微镜进行活细胞成像。最常见的显微镜落射荧光广角和纺纱盘共聚焦。在这里，使用具有200瓦特金属卤化物光源的自动化和电动广角落射荧光显微镜。 </li><li>获取舞台顶部或显微镜环境舱。在这里，使用一个阶段-T运算环境舱。 </li><li>使用商业软件与实用程序运行显微镜来执行时间推移显微镜。 </li><li>使用市售的软件或ImageJ的图像分析。许多其他分析工具是市售的，并有许多已发表​​8,18的定做的程序。 </li></ol> 2.蜂窝可视化流程和表型响应<ol><li>可视化与明显微镜细胞。单独微分干涉对比，相位相反可以很翔实，研究抗癌药物反应的反应。这些过程可包括有丝分裂，细胞运动和细胞凋亡。 </li><li>可视化的细胞结构，细胞器和流程荧光生物传感器。荧光探针是跟踪特定的亚细胞的过程信息。这些可以包括微管动力学，线粒体，内质网动力学​​，蛋白质积累和本地化，以及分子信号（ 例如 ，磷酸化，钙）。 </li></ol> 3.样品的制备<ol><li>生长细胞在细胞培养认证菜肴在一个标准的，加湿细胞培养孵化器（ 例如，37℃，5％CO 2，相对湿度80％）。 <ol><li>成长HT1080细胞与EBSS MEM。补充媒体有10％v / v的FBS，1％体积/体积青霉素/链霉素，1％体积/体积丙酮酸钠和1％体积/体积的非必需氨基酸。 </li></ol></li><li>成像之前两天，板50000 HT1080 FUCCI细胞变成在认证的无菌层流罩12孔＃1.5玻璃底皿的3个孔。调整镀达到〜60％汇合的实验开始细胞的数量。取决于细胞系和实验的细胞密度的性质可能更少。 注:细胞密度可能对细胞系的生长和对实验结果深刻的影响。计数细胞，以尽量减少实验对实验variabilit由于密度年。 <ol><li>根据环境室和用于实验的必要条件，板的细胞在单个孔培养皿（通常35-或60毫米），4-以及35毫米的菜肴，6-，12-或24孔细胞培养板，或盖玻片在各种格式的幻灯片。通过细胞培养塑料使用带有＃1.5玻璃作为最物镜是此厚度玻璃的优化，和成像玻璃底板是非常差。 注意:某些细胞系不成长，在玻璃上生存。在这些情况下，玻璃可以以增强细胞粘附进行包衣（ 例如 ，多聚赖氨酸或胶原蛋白）。一些公司制造光学细胞培养塑料，它必须根据经验来确定，如果这是一个可行的选择。 </li></ol></li></ol> 4.环境商会建立<ol><li>之前的任何实验，建立了环境室，以便它工作在〜80％ ​​的湿度，因此在样本位置的温度为37℃。大多数培养的细胞生长，在5％ 的 CO 2 /平衡空气气氛由于在介质碳酸氢钠缓冲液。根据设置，或者设置环境控制器，5％CO 2，这将100％的CO 2与空气混合，或使用预先混合，认证，5％CO 2 /平衡空气的气体。按照气体的流速制造商的说明。 注意:某些细胞系生长中的CO 2独立介质在这种情况下，它们可以在没有CO 2被维持。专门设计的成像介质也可以限制附加自发荧光化合物。增长论文介质所需的细胞类型必须凭经验事先确定。 </li><li>成像之前，确保水贮存器被用无菌蒸馏水填充（按照生产商的指示）。打开环境室到所需的设定温度，并将其放置到显微镜阶段插图。为了节省汽油，不开始在这一点上流动。 注意:如果在舞台顶部室和舞台插图和/或连接线将可能会引入运动伪影到实验室任何紧张之间的自由空间。 <ol><li>莱石蜡的影片片段在之前室室插入它耦合到舞台的舞台插入口边缘，并确保没有连接拉动室。 </li></ol></li><li>允许室平衡至37℃。保持稳定的温度，以防止成像这可能会影响细胞的生理和引进漂移过程中的温度波动。达到温度平衡通常需要30分钟到1小时，这取决于环境。 <ol><li>插入带有水的井一个"虚拟"的菜进入室中，同时升温。成像盘填充水模拟物样品，有助于确保该室被充分加热，并稳定化。填充预温无菌水室减少了所需的对温度稳定的时间和最小化在加入实验样品的温度降低。 </li></ol></li></ol> 5.显微镜建立<ol><li>打开显微镜，相关联的计算机和任何所需的外围设备。 <ol><li>根据不同的光源，迫不及待地打开它，直到需要（ 例如 ，LED）。 </li></ol></li><li>在较低的位置的客观炮塔，选择20X 0.7 NA目的使用。 </li><li>在目标样本位置 - 这将使它更容易找到细胞时，样品在室内。 </li><li>此时限定，如果它们是从以前的实验已知的成像参数。 </li></ol> 6.运送到细胞显微镜和成商会<ol><li>必须从孵化至环境室成像输送细胞。确保，这是迅速完成，以限制相对低的CO 2的影响</子&gt;的气氛和对细胞减少温度。按照样品运输和清理的泄漏情况下，所有的机构的生物安全指南。 <ol><li>将在一个密封的发泡胶容器中或隔热袋细胞避免大环境的变化。 </li></ol></li><li>插入以下制造商的指示将样品成像盘进入腔室。 </li><li>所述细胞已经在环境室之内固定后，密封所述腔室，以保持一个稳定的环境，并立即转气氛气体的源（S）上。 <ol><li>作为一些环境试验箱不利用紧，密封的盖子，以阻止在生长培养基的顶部蒸发，层无菌，胚胎认证矿物油。裹气透性薄膜石蜡样品周围的菜小心不要阻碍玻璃成像区域的周界边缘。本地化，仲加湿方法可以采用。凝结在样品上盘可减少perfor的盖子明显微镜曼斯。 </li></ol></li><li>为了最大限度地减少在实验过程中早期的热漂移的影响，等待30分钟开始的时间推移之前。在规定的时间变化的基础上成像盘的大小和其他因素。凭经验判断。 </li></ol> 7.设置成像<ol><li>选择成像条件，将最能代表该数据。采取预防措施，通过限制曝光时间，使用低强度光线和选择是由光的波长更长兴奋探头，以避免光毒性。 </li><li>每个位置限定x，y和z平面和期望波长（多个）被成像。时间分辨率是由位置和波长的数量的限制。对于大多数细胞过程的长期时间推移，获取每10一个图像 - 20分钟;较高的时间分辨率（短的间隔， 例如 ，1分钟）提供了更多的数据点，并因此更坚固细胞跟踪，也导致更多的集成曝光，更大的数据集。 <ol><li>由于HT1080 FUCCI具有动态绿色和红色荧光蛋白（见代表性的结果），请使用以下的曝光时间:FITC / GFP - 50毫秒，德州红/ TRITC - 40毫秒，明场 - 20毫秒。使用2×2箱。 </li></ol></li><li>使用在高级设置的默认参数启用软件控制自动对焦。限定为10微米的自动聚焦范围与建议的步长。请务必在启动延时之前做到这一点。与明图像自动对焦，并从不与荧光，减少光毒性和光漂白。 <ol><li>使用与电动载物台显微镜的任何软件控制的自动对焦系统，并直接着眼于样本。然而，它们可以限制实验的采集速度。硬件控制的连续聚焦系统时间推移显微镜工作良好，提高速度，从而允许更多的位置被成像或获取更高的速度。然而，他们依靠检测空气 - 玻璃界面和可能失去样品焦点在整个井玻璃厚度变化。 </li></ol></li><li>与包含已加热到37 摄氏度所需的药物浓度介质更换一半的媒体部分媒体更换有助于减少热漂移。在这个实验中的条件是载体（DMSO），1μM的selinexor和10μMPD0332991。 注意:此步骤也可以开始通过暂停和重新启动的实验收购后完成。这允许单个细胞的前，后的药物反应纵向跟踪。如果药物稳定性或代谢是一个问题，暂停和更换媒体可以用相同的方法来完成。为了测试药物降解或代谢，从处理的细胞的介质也可以被放置在幼稚细胞来测试药物作用。 </li><li>开始的时间推移。 </li><li>随着时间的推移运行，确保成像领域保持重点和chambe- [R保持湿润37℃。 <ol><li>根据需要按时间点之间的时间间隔期间暂停收购调整焦距。 </li></ol></li><li>如果有必要，在长时间实验中加水至室内。避免在37℃。加入预热，无菌蒸馏水冷却室</li></ol> 8.结束时间推移<ol><li>当实验运行完毕，停止收购，如果它没有被设置为自动停止。 </li><li>确保时间推移已经被正确地保存到硬盘驱动器，虽然大部分软件程序包在采集过程中自动保存的话还没说完正确的数据可能会丢失。 </li><li>从显微镜中取出室和细胞处理成以下机构批准的生物安全程序的生物危害性的废物。 </li></ol> 9.纵向跟踪和分析时间推移数据<ol><li>选择方法进行分析是适当感兴趣的生物过程。对ImageJ的许多插件，使用MATLAB程序，以及自定义平台已经产生了特定的应用程序。以下方法涵盖细胞核和核荧光探针的分析的跟踪如在图4 和 5证实。 </li><li>打开ImageJ的中所利用的渠道。TIF图像堆栈。另外，使用该插件Bioformats直接在ImageJ的收购方案打开本机文件。 </li><li>一个小区的使用明视野图像或核标签通道（如果使用的话）的细胞核内绘制的感兴趣的区域，并把它添加到的ROI经理。注意:如果成像细胞有核标签（ 例如 ，组蛋白H2B-EGFP），然后自动跟踪的细胞可利用通过时间来产生利益（投资回报）表示各个核的区域。 </li><li>进行到下一个时间点，并且在同一胞核内的ROI的位置秒。投资回报率增加了投资回报率经理。 </li><li>继续跟踪和使感兴趣区为单个细胞，直到有一个蜂窝事件（有丝分裂，细胞凋亡等 ）或细胞不再能够跟踪（ 即 ，移出帧的或在实验结束时）。 </li><li>当感兴趣区已经建立了通过它们在该领域的时间单元，叠加他们到感兴趣的荧光通道。测量每个信道的每个时间点上的荧光的平均强度。保存以备后用ROI列表。 <ol><li>各种测定可在ROI内进行为在不同的实验程序。点击分析→设置测量...弹出一个菜单，选择的分析方法（ 例如 ，指的是投资回报率，综合强度等内强度）。 </li></ol></li><li>注意单个细胞，同时跟踪细胞命运（ 如 ，细胞凋亡，分工，生存）。这允许创建存活曲线和furthe的- [R人口分析。 </li><li>随着两个通道的平均亮度，营造散点图响应治疗（ 图5B，C），以显示细胞周期的动态。 注意:图解可以为单个细胞被创建随时间或平均的跟踪的细胞群。定量成像可用于响应于癌症治疗建立复杂的反应和细胞的关系是至关重要的。长期时间推移显微镜，纵向跟踪和数据分析是一个多步骤的过程中，随着对的显微镜和分析工具的类型很多选项，并遵循图1中所提供的一般轮廓。 </li></ol>

The authors have no conflicts of interest to disclose.

时间推移显微镜和纵向跟踪的优势 该显微镜是用于药物反应的纵向研究的理想工具，因为它允许调查者跟踪单个细胞和他们的命运以及整个人口。变异的细胞群内的药物反应是用于抗癌治疗设计的一个主要问题。单个细胞的纵向跟踪调查可以观察到这种变化，并开始了解的底层机制和后果，因为它涉及到细胞群。利用各种荧光探针提供的方法来观察和了解双方?...

活细胞显微镜和单细胞的纵向跟踪不是一个新的技术。从最早的显微镜，爱好者和科学家们观察和研究单个细胞和生物体，他们的行为和发展1-3。从已故的大卫·罗杰斯在20世纪50年代范德比尔特大学一个著名的例子显示了血涂片追金黄色葡萄球菌和吞噬4的最终过程中的人中性粒细胞。此活细胞电影是如何多个进程可以观察和相关的单次实验优异的例证:化学梯度，力学和细胞运动的速度的感测，细胞形状动力学，粘附和病原体的吞噬作用。 全自动显微镜和高度敏感的数码相机的出现，导致了使用显微镜调查的越来越多问细胞生物学范围˚F基本问题ROM细胞如何移动和5,6分7,8细胞器的动态和膜贩运9-11。无荧光，明场显微镜，包括相位对比（PC），它囊括了诺贝尔奖为弗里茨·泽尔尼克于1953年，和微分干涉对比（DIC）允许细胞和细胞核，而且亚细胞结构，包括微管束的观察，染色体，核仁，细胞器动力学和粗纤维肌动蛋白12。基因编码荧光蛋白和细胞器对荧光染料的发展已经大大影响时间推移显微镜13-15。尽管本文的不关注的焦点，在细胞球体和在使用共聚焦和多光子显微镜原位 （活体显微镜）成像代表了该方法的另一个扩张，并有使用和讨论这些方法16-19优秀物品。 细胞的抗CANC响应呃药物或天然产物的分子和细胞比例来确定。理解细胞应答和命运以下治疗常常涉及人口平均测定（ 例如 ，免疫印迹，全井措施），或与免疫荧光检测固定时间点和流式细胞仪，用来测量单细胞。异质性群体内单个细胞应答的药物，特别是在肿瘤中，可以解释一些变异的响应跨越了与在饱和相同药物处理的细胞系和肿瘤中看到。长期纵向方法遵循给定的单细胞或细胞群是不常见的，但非常强大的方法，其允许分子响应途 ​​径的直接研究，不同表型（ 例如 ，细胞死亡或细胞分裂），观察细胞至细胞变异的群体内，以及如何将这些因素有助于人口响应动力学20-22。乐观，能够观察和量化的单细胞反应将有助于改善我们的药是如何工作的，为什么他们有时会失败，以及如何最好地使用他们的理解。 长期时间推移显微镜，纵向跟踪，以及药物反应分析的技术可用于许多研究者，并且可以是简单的，只用透射光观察的表型反应20,21。该方法的主要组成部分包括:感兴趣的细胞的适当的制剂，用环境室的自动显微镜，照相机与计算机集成的获取和存储图像，以及软件审查时间推移和测量和分析细胞和任何荧光生物传感器。我们提供了一个详细的协议，它为使用明和/或广角镜啶进行培养细胞的时间推移显微镜，只要几天许多技巧。该协议可以用于任何细胞系，可以在培养物中生长研究其抗癌治疗的反应。我们提供获得的数据的实施例和使用多个不同的基因编码的荧光生物传感器和相衬显微镜的一例，简要地讨论的不同类型的探针，优点和长期的时间推移和纵向跟踪的缺点进行了分析，什么都可以了解到这种方法，很难从非直接方法，以及一些变化，我们希望将有兴趣和价值经验的研究人员使用的方法谁没有考虑到理解，以及经验丰富的研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Taxol (paclitaxel)</td>
			<td>Sigma</td>
			<td>T7191</td>
			<td>microtubule stabilizing drug</td>
		</tr>
		<tr>
			<td>etoposide</td>
			<td>Selleckchem</td>
			<td>S1225</td>
			<td>topoisomerase II inhibitor</td>
		</tr>
		<tr>
			<td>selinexor</td>
			<td>Karyopharm Therapeutics</td>
			<td>na</td>
			<td>XPO1/CRM1 inhibitor, gift</td>
		</tr>
		<tr>
			<td>Kinesin-5 inhibitor</td>
			<td>Merck Serono</td>
			<td>na</td>
			<td>gift, also available from American Custom Chemicals Corporation. CAS 858668-07-2</td>
		</tr>
		<tr>
			<td>cell growth medium</td>
			<td>HyClone (Fisher) or Mediatech</td>
			<td></td>
			<td>many companies available</td>
		</tr>
		<tr>
			<td>5% CO2/balance air, certified</td>
			<td>Airgas</td>
			<td>Z03NI7222004379</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm dish, 20mm glass bottom</td>
			<td>Cellvis</td>
			<td>D35-20-1.5-N</td>
			<td>many companies available</td>
		</tr>
		<tr>
			<td>35mm 4 well dish, 20mm glass bottom</td>
			<td>Cellvis</td>
			<td>D35C4-20-1.5-N</td>
			<td>many companies available</td>
		</tr>
		<tr>
			<td>35mm dish, gridded glass bottom</td>
			<td>MatTek</td>
			<td>P35G-2-14-CGRD</td>
			<td>many companies available</td>
		</tr>
		<tr>
			<td>multi-well, glass bottom</td>
			<td>Cellvis</td>
			<td>P12-1.5H-N</td>
			<td>many companies available</td>
		</tr>
		<tr>
			<td>Olympus IX81 inverted epifluorescence microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX2-UCB controller</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PRIOR LumenPro200</td>
			<td>Prior Scientific</td>
			<td>Lumen200PRO</td>
			<td></td>
		</tr>
		<tr>
			<td>PRIOR Proscan III motorized stage</td>
			<td>Prio Scientific</td>
			<td>H117</td>
			<td></td>
		</tr>
		<tr>
			<td>STEV chamber</td>
			<td>InVivo Scientific</td>
			<td>STEV.ECU.HC5 STAGE TOP</td>
			<td></td>
		</tr>
		<tr>
			<td>Environmental Controller Unit</td>
			<td>InVivo Scientific</td>
			<td>STEV.ECU.HC5 STAGE TOP</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu ORCA R2 CCD with controller</td>
			<td>Hamamatsu</td>
			<td>C10600</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon laser launch</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SOLA light engine</td>
			<td>lumencor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iXon Ultra 897 EM-CCD</td>
			<td>ANDOR&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TOKAI HIT inclubation chamber</td>
			<td>TOKAI HIT</td>
			<td>TIZSH</td>
			<td></td>
		</tr>
	</tbody>
</table>

through the looking glass: time-lapse microscopy and longitudinal tracking of single cells to study anti-cancer therapeutics

单细胞对抗癌药物的响应中确定人口响应显著贡献，并因此是在整体结果的主要因素。免疫印迹，流式细胞术和固定细胞实验经常用于研究细胞的抗癌药物的反应。这些方法是重要的，但是它们具有若干缺点。变异药物反应癌细胞与正常细胞之间的，和瞬态响应罕见的癌症不同来源的细胞，之间是很难理解用人口平均测定，不能够直接跟踪和纵向分析它们。显微镜特别适合于图像的活细胞。技术的进步使我们能够在一个分辨率不仅能细胞跟踪，而且多种细胞反应观察常规图像的细胞。我们详细描述了一种方法，允许的连续时间推移成像为基本上只要需要，典型地高达96小时的药物的反应过程中的细胞。使用该方法的变型中，细胞可以为周进行监测。随着就业的基因编码荧光生物传感器大量的进程，途径和应答可以遵循。我们表明的例子，其中包括跟踪和细胞生长和细胞周期进程，染色体动力学，DNA损伤的定量，和细胞死亡。我们还讨论技术和灵活性的变化，并突出显示一些常见的陷阱。

长期时间推移显微镜和直接纵向跟踪允许许多抗癌作用药物的反应在研究。以下图1中的大致轮廓，示出细胞的多个例子表达验证荧光记者，具有抗癌药物，追踪处理，并使用不同的方法进行分析。 单独相衬显微镜是非常翔实和有力的间期与有丝分裂，有丝分裂期和停滞，不正常的细胞分裂和细胞死亡21,23,24</su...

Watch this Scientific Journal Video about Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics at JoVE.com

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

透过玻璃看:时间推移显微镜和单细胞的纵向跟踪研究抗癌治疗

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

through-looking-glass-time-lapse-microscopy-longitudinal-tracking

Research

JoVE Journal

Medicine

10.9K 次观看.  University of Colorado, Boulder. Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

视频: Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

新型抗癌药物使用手术标本肿瘤植的发展方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

科研

医学

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

测量和制止在弱视治疗

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

清醒小鼠滴鼻的CNS药物治疗管理

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive methods1.&#160;Subjects are placed in the supine position and allowed to rest quietly for at least 10 min prior to the start of the exam. The proper cuff size is selected and a blood pressure is obtained using an oscillometric device. Once a resting blood pressure has been obtained, pressure waveforms are acquired from the right femoral and right common carotid arteries. The system then automatically calculates the pulse transit time between these two sites (using the carotid artery as a surrogate for the descending aorta). Body surface measurements are used to determine the distance traveled by the pulse wave between the two sampling sites. This distance is then divided by the pulse transit time resulting in the pulse wave velocity. The measurements are performed in triplicate and the average is used for analysis.

Pulse wave velocity (PWV) measurement assesses arterial stiffness by a tonometry-based system that measures the speed with which arterial pressure wave travels along the arterial tree, typically approximating the time that it takes this wave to travel from the descending aorta (using the carotid artery as a surrogate) to the femoral artery. Carotid-femoral PWV increases two- to threefold across the adult lifespan.

脉搏波传导速度测试老化的巴尔的摩纵向研究中

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

焦时间推移成像为牙科复合材料的细胞相容性评价的有效方法

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.