Name: the use of ex vivo whole-organ imaging and quantitative tissue histology to determine the bio-distribution of fluorescently labeled molecules
Uploaded: 2016-12-24T15:00:00.000+00:00
Duration: 8 min 7 s
Description: Watch this Scientific Journal Video about The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules at JoVE.com

Partial salary support for GLB is provided by NIH grant R01HL121527. JWDM is supported by the Currier Fellowship in Neurology.

注:在本协议中的所有动物的使用是经密西西比医学中心大学的机构动物护理和使用委员会。 1.动物和组织处理<ol><li>管理和牺牲<ol><li>麻醉，用3％异氟烷将动物5分钟，并通过观察不存在脚趾捏反射的验证麻醉深度。应用兽医软膏眼睛防止干燥时的麻醉下。 </li><li>称重动物并吸取或罗丹明标记的ELP，SYNB1-ELP，或达-ELP的50毫克/公斤的剂量为1.5毫升管中。减100微升施用标记的化合物用于后续步骤的标准曲线的制剂。 注意:重要的是，标准从同一批次向动物给药标记的蛋白质的，以避免在荧光标记效率由于变性的差异进行。为了正确地去除背景fluores从后荧光测量萤光，未处理的动物必须使用相同的方法/试剂作为治疗的动物被处死。 </li><li>管理与使用任一尾静脉或股静脉（其通过1厘米的切口在腹股沟访问）先前描述1的IV注射。镇痛，如果制作腹股沟切口，辖卡洛芬皮下剂量为5毫克/公斤，并与伤口夹子闭合之前申请sensorcaine到切口部位。要通过鼻内（IN）航线管理，将动物在仰卧位。 </li><li>使用2μL移液器，制定标记化合物的1μL下降。简言之，将动物的头从麻醉衣领滑动以允许访问鼻孔。 </li><li>吸移管的尖端放置到单个鼻孔的开口，慢慢压下柱塞，从而允许所述液滴向下运行的鼻孔进入鼻腔。重复每1分钟，交替鼻孔每一滴直到全部剂量广告服侍。 注意:通过在管理的最大音量不得超过10 - 15μL鼠。 </li><li>他们每移动到一个单独的笼子前，留在仰卧位动物麻醉下进行10分钟的最后给药后。 注意:不要离开无人看管的动物，直到他们重新获得足够的意识，以保持胸骨斜卧。 </li><li>给药后1小时，如之前重新麻醉的动物，并使用PBS（210.0毫克/升KH 2 PO 4，9,000.0毫克/升氯化钠，726毫克/升的 Na 2 HPO 4·7H 2 O）在经由穿心灌注牺牲他们10毫升/分钟，总共20毫升。 注意:动物在处死灌注以从组织中移除所有的血液需要确定的标记的化合物的血管外的浓度。灌注试剂可导致改变的背景荧光，并应保持一致。牺牲的方法应该是一致的内的组。牺牲的不同方法会导致在脉管内变化的血液水平，改变了组织的背景荧光。 </li></ol></li><li>组织集合<ol><li>如上述用于漂洗除去组织描述制备PBS中。 注意:如果另一个缓冲器被用作灌注液，在相同的缓冲液冲洗组织。 </li><li>通过简单的夹层中取出心脏，肺，胃，肝，脾，肾。在PBS溶液清洗使用实验室之前湿巾抹干或吸走多余的水分，并把组织上的成像垫子。 </li><li>斩首鼠标和使用刀具骨或骨钳去除头骨的顶部。使用镊子，小心地取出大脑，保持嗅球完好。冲洗，干燥，并将其放置在所提供的影像垫。 </li><li>要通过快速弹射7去除脊髓，填充10毫升的注射器，用PBS和附加迟钝18号针头。 注:18号适合许多鼠标线的成人椎管。所需的直径将动物大小而异。 </li><li>置于俯卧位的动物，以确保椎管在断头站点的开口被干净地切割和自由的血液。 </li><li>来自各地的脊柱去除皮肤。使用锋利的刀骨或剪刀，通过在腰节直接喙臀部脊柱切断。 </li><li>简单地说，用PBS冲洗可视化椎管。轻轻插入针头进入椎管。 注:针应该感到舒适，并可能需要一些缓慢滚动的插入过程中来回穿梭。如果针松的感觉，更切喙可作出或可能需要较大的直径针。 </li><li>用拇指和食指，将压力施加到插入针周围的脊柱。按下中度和恒压活塞。脊髓将弹出完整出喙开放的脊髓柱。 注:彻底清除，冲洗，以及所有主要器官可以在5合理完成烘干 - 7分钟。这样短的时间内阻止对PBS扩展存储，这可能会导致过度曝光，并从组织标记的蛋白质浸出的需要。 </li></ol></li></ol> 2.全器官离体荧光成像<ol><li>成像参数<ol><li>启动图像采集的软件。在收购控制面板的右下方，点击"初始化"。 </li><li>在"成像模式"，选择"荧光"复选框。在"曝光时间"，选择"自动"。 </li><li>在"分级"，选择"小"和F /停止设置为2。 </li><li>选择535 nm激发和580 nm发射过滤器。 注意:这将取决于所使用的标签上会有所不同。 </li><li>选择"视图B场"，并设置圆盾ŧ高度0.3厘米 注意:这将取决于组织被成像而变化。 </li><li>一旦温度指示器变为绿色（意味着机器完全初始化），将组织到提供黑色背景垫，从而确保组织被完全彼此分离。放置在成像器的垫，以确保所有的组织都在所选区域的网格内。较大的和非均质的组织，如全肝，应布置，以便减小波瓣重叠。 注意:组织可以一起在单个组被布置，使一个图像，以容纳一单个动物或相似类型的所有组织中。这使得更容易稍后的分析和组织。如果在荧光强度大的差异是各种组织中本，最好是图像它们单独或作为同一组织类型的基团，作为一个单一的曝光时间可能不适合于所有的组织的强度。 </li><li>点击"获取"。 </li><li>成像后，立即取出组织并存储或冻结它们定量组织学，如在步骤4中概述。 </li></ol></li></ol> 3.标准和图像处理<ol><li>标准<ol><li>在PBS中，创建在动物用于治疗，开始于原始浓度各蛋白质的两倍连续稀释，以总共15稀释液。 注意:浓度范围应该旨在包括预期的组织浓度，并可能需要与原始样品的附加的稀释，这取决于起始浓度。 </li><li>吸取100μL的每个标准成黑色的96孔板的每个孔中的。包括纯PBS中的一个孔中，以获得背景荧光的测量。 </li><li>图像与用于组织成像相同的参数标准。 </li><li>在图像采集软件"工具选项板"下的"投资回报工具"，选择"感兴趣区域（ROI）"圈子，并绘制一个井周围的投资回报率。复制ROI所有其他填充井，然后点击"衡量投资回报率。" </li><li>减去在从其他的标准值的纯PBS中平均辐射效率的值;这消除了背景荧光。上绘制散点图对已知浓度值。 注意:得到的线性趋势线的方程可以被用于计算相对荧光值。 </li></ol></li><li>图像处理<ol><li>在图像采集软件，选择自由ROI工具，绘制的ROI周围每个独立的组织。点击"措施"。 </li><li>用所得平均辐射效率的测量，减去未处理动物的每个组织类型测量的值和先前创建标准曲线上绘制这些值。 注意:得出的相对荧光值提供的快速，准确的快照标记化合物的生物分布。 </li></ol></li></ol> 4.定量组织组织学<ol><li>对切​​片的制备和冷冻的组织<ol><li>创建或购买足够的尺寸以冻结所选组织和低温恒温器的容器中使用。周围适当尺寸和形状的物体包裹铝箔效果很好。 </li><li>通过填充一个500ml的烧杯中的一半用干冰和加入异丙醇的〜350毫升，以便有足够的液体的干冰以上以部分地浸没冷冻容器准备异丙醇和干冰的混合物中。 注意:液氮经常导致最佳切割温度化合物（OCT）和包埋组织的断裂，应该避免。 </li><li>与华侨城部分填充冷冻集装箱，确保有不存在泡沫。如果有气泡，使用镊子工作气泡从溶液或类似的工具。 </li><li>除去组织从PBS中并干燥它彻底，或者用一个实验室擦拭或通过触摸细腻组织到实验室的边缘擦拭和允许液体吸走轻轻拍打它。 </li><li>轻轻地放在组织到华侨城的冷冻集装箱内，确保不引入气泡组织下方。一旦组织就位，添加OCT中直至组织被完全覆盖。 </li><li>浸没冷冻集装箱尽可能是可能的，而不使异丙醇进来与暴露十月接触的容器内。允许30 - 90秒（或可能更多更大的组织）的OCT和组织在-80℃保存，只要需要之前完全冻结。 </li></ol></li><li>标准的制备切片。 <ol><li>准备并通过去除筒的端部正确标记1毫升一次性使用的注射器，使得所述注射器的整个长度为一个直径。这将允许冷冻汽缸后面将要推出。 </li><li>我们ING OCT中，创建施用的化合物的15步两倍系列稀释，如用PBS先前完成的，但用1 mL的最终所得的体积。包括用于去除背景荧光值纯OCT的一个样本。 </li><li> OCT的粘度使得充分混合困难。反转试管，华侨城再次反转之前，彻底解决。重复此10 - 20次，以确保一均匀的混合物。混合过程中，使标准溶液在4℃和避光。 </li><li>混合后，再次准备异丙醇和干冰的混合物中，如前。 注意:液态氮也可以代替异丙醇/干冰混合物的使用，只要它们冻结固体后注射器迅速取出。 </li><li>绘制OCT标准入注射器，同时注意引进尽可能少的气泡越好。拟定OCT溶液后，立即直接浸注射器插入异丙醇，并允许该溶液完全冻结20秒 - 注射器10中。存储注射器在-80℃下，只要必要的。 </li></ol></li><li>组织和标准的切割<ol><li>将两种组织和标准-20°C和足够的时间让所有样品来温度。 注:理想的切削温度一般落在周围的-10°C范围内对大多数组织中，但它最终取决于组织的大小和脂肪含量必须凭经验细化。 </li><li>使用低温恒温器，切割组织成20微米厚的切片，并将其安装到滑动，如前所述8。在-20℃保存幻灯片和切割过程中，直到扫描使他们免受光线尽可能远。 注意:重要的是在这里，要测量的部分在整个组织的整个厚度被适当地分布。 </li><li>要安装到标准的低温恒温器吸盘，采取从冷冻注射器和温暖外3 - 5秒通过霍尔鼎在手里。推动柱塞，直到在OCT气缸的短节（约1厘米）是注射器的外部。 <ol><li>用剃刀刃，切通过气缸和垂直放置所得汽缸部上的卡盘，使用OCT以保持它在适当位置。这种构造应该导致良好定义的圆圈被切断。 </li></ol></li><li>切片气缸20微米，从每个稀释放置一个片到单个滑动，使一个滑动表示整个标准曲线。储存在-20℃下将载玻片直到准备扫描。 </li></ol></li><li>扫描和定量<ol><li>启动扫描阵列软件，点击"543激光"开始激光启动过程。等待15分钟的激光做好准备。 </li><li>一旦激光准备就绪时，从-20取出幻灯片℃，并将它们移动到一个大的，光保护的容器来的温度和干燥3分钟。这将防止受潮在SLI德扫描仪。 </li><li>加载滑入上滑动扫描器滑动容器。点击"扫描"，选择"运行轻松扫描。" </li><li>设置扫描分辨率为50微米。 </li><li>在"荧光团"选择第一个"使用"复选框，并选择"TRITC"设置PMT增益为80％。 注意:这些设置将取决于荧光团和标记分子的浓度变化。 </li><li>在左手侧，确保了扫描区域包括整个幻灯片。点击"开始"。 </li><li>扫描后，选择"文件"，"另存为"，然后将图像保存为.tif。 注意:组织和标准的幻灯片应在相同的设置进行扫描。 </li><li>加载生成的图像转换成ImageJ的，选择投资回报组织和标准各地。在"分析"选项卡中，选择"设置测量"，选择"平均灰度值。"点击220;措施"。 注意:与前面的技术中，背景荧光必须从所有测量值中减去。 </li><li>从绘制对已知浓度的标准平均灰度值测量，以创建标准曲线。 注意:从身体组织将所得的测量应在每个组织内进行平均并绘制该标准曲线上，以确定在组织内的浓度。 </li></ol></li></ol>

<ol>
	<li>George, E. M., Liu, H., Robinson, G. G., Bidwell, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Polypeptide+Drug+Carrier+for+Maternal+Delivery+and+Prevention+of+Fetal+Exposure.">A Polypeptide Drug Carrier for Maternal Delivery and Prevention of Fetal Exposure.</a> J. Drug Target. 22 (9), 1133-1145 (2014).</li><li>George, E. M., Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factor+purification+and+delivery+systems+(PADS)+for+therapeutic+angiogenesis.">Growth factor purification and delivery systems (PADS) for therapeutic angiogenesis.</a> Vasc. Cell. 7 (1), 1-11 (2015).</li><li>Bidwell, G. L., Perkins, E., Hughes, J., Khan, M., James, J. R., Raucher &amp;amp, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermally+Targeted+Delivery+of+a+c-Myc+Inhibitory+Polypeptide+Inhibits+Tumor+Progression+and+Extends+Survival+in+a+Rat+Glioma+Model.">Thermally Targeted Delivery of a c-Myc Inhibitory Polypeptide Inhibits Tumor Progression and Extends Survival in a Rat Glioma Model.</a> PLoS One. 8 (1), (2013).</li><li>Liu, H., Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+improved+blood&#8211;brain+barrier+permeability+of+endomorphin-1+using+the+cell-penetrating+peptide+synB3+with+three+different+linkages.">The improved blood&#8211;brain barrier permeability of endomorphin-1 using the cell-penetrating peptide synB3 with three different linkages.</a> Int. J. Pharm. 476 (1-2), 1-8 (2014).</li><li>Flessner, M. F., Fenstermacher, J. D., Blasberg, R. G., Dedrick, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peritoneal+absorption+of+macromolecules+studied+by+quantitative+autoradiography.">Peritoneal absorption of macromolecules studied by quantitative autoradiography.</a> Am. J. Physiol. 248, 26-32 (1985).</li><li>Flessner, M. F., Choi, J., He, Z., Credit, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+characterization+of+human+ovarian+cancer+cells+in+a+rat+model+of+intraperitoneal+antineoplastic+therapy.">Physiological characterization of human ovarian cancer cells in a rat model of intraperitoneal antineoplastic therapy.</a> J Appl Physiol. 97, 1518-1526 (2004).</li><li>Kennedy, H. S., Jones, C., Caplazi, P., Iii, C. J., Caplazi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+standard+laminectomy+with+an+optimized+ejection+method+for+the+removal+of+spinal+cords+from+rats+and+mice.">Comparison of standard laminectomy with an optimized ejection method for the removal of spinal cords from rats and mice.</a> J. Histotechnol. 36 (3), 86-91 (2013).</li><li>Currle, D. S., Monuki, E. S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+Freezing+and+Cryosectioning+E12.5+Mouse+Brain.">Flash Freezing and Cryosectioning E12.5 Mouse Brain.</a> J. Vis. Exp. JoVE. (4), e198 (2007).</li><li>Walker, L., Perkins, E., Kratz, F., Raucher &amp;amp, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+penetrating+peptides+fused+to+a+thermally+targeted+biopolymer+drug+carrier+improve+the+delivery+and+antitumor+efficacy+of+an+acid-sensitive+doxorubicin+derivative.">Cell penetrating peptides fused to a thermally targeted biopolymer drug carrier improve the delivery and antitumor efficacy of an acid-sensitive doxorubicin derivative.</a> Int. J. Pharm. 436 (1-2), 825-832 (2012).</li><li>Frangioni, J. V. <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+near-infrared+fluorescence+imaging.">In vivo near-infrared fluorescence imaging.</a> Curr. Opin. Chem. Biol. 7 (5), 626-634 (2003).</li><li>Vugmeyster, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+toxicology+of+therapeutic+proteins:+Advances+and+challenges.">Pharmacokinetics and toxicology of therapeutic proteins: Advances and challenges.</a> World J. Biol. Chem. 3 (4), 73 (2012).</li><li>Stumpf, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+and+microscopic+autoradiography+to+determine+tissue+distribution+of+biopharmaceuticals+-+Target+discoveries+with+receptor+micro-autoradiography+engendered+new+concepts+and+therapies+for+vitamin+D.">Whole-body and microscopic autoradiography to determine tissue distribution of biopharmaceuticals - Target discoveries with receptor micro-autoradiography engendered new concepts and therapies for vitamin D.</a> Adv. Drug Deliv. Rev. 65 (8), 1086-1097 (2013).</li></ol>

GLB is owner of Leflore Technologies, LLC, a private company working to develop ELP-based drug delivery technology. Leflore Technologies provided no support for the publication of this paper or for the generation of data within it.

而离体全器官成像一般是直接的，粘附到一些基本概念和技术可以改善生物分布测量的准确度。的光的经验短的波长在大多数组织中高度的散射和吸收的，这显著影响短波长的荧光团的效用。这些荧光限制了深部组织研究中的应用，但他们已在实验中有效地用于寻找表面组织或进入眼内。相反，光的长波长经受显著较少的散射和吸收，造成更大的渗透和具有较厚组织10更准确的结果...

荧光标记是用于确定化合物的生物分布，使用一系列的设备是共同的许多实验室一个容易使用的，有效的方法。荧光团是广泛可用的，相对便宜的，以及各种波长，使得多个标记分子可以同时使用无干扰的进入。大多数荧光团具有一系列化学缀合到目标化合物不同的活性基团的，和缀合的过程通常是简单的为大多数类型的反应性位点的。此外，对于荧光标记的化合物的测量所需的设备在许多实验室是常见的。荧光显微镜，成像仪，和滑动扫描器都可以在不同的情况下使用，使得使用荧光标记的高度可访问的。荧光标记经常用于确定在体内和离VIV化合物的生物分布和动力学Ø与实时成像设备，如IVIS光谱成像仪，并通过定量组织组织学2,3。 离体全器官成像的使用实时成像设备的使用增加了随着时间的推移，由于其易于使用和快速创建标记的化合物的相对浓度的准确的比较，而无需tissues4的进一步处理能力。然而，虽然离体全器官成像可以允许容易分析和比较，它不产生一个组织内绝对化合物浓度的定量测量。这是由于完整的器官内的光散射的影响。由于光散射（以及在较小程度上，吸光度）由组织的大小和密度而变化，整个器官成像能够低估在大型或密集的器官组织水平。制定相应的标准绝对浓度测量也很困难，因为我们必须模仿厚度和每个器官的密度。另一方面，整个器官成像获得的试剂的相对组织水平的快速方法，它是理想的比较多个相关分子的相对生物分布（例如，在药物靶向研究）。一种替代的策略是利用荧光定量组织学，从定量放射自显影的方法中派生的技术，以获得测试试剂5,6-绝对组织水平。而不是将整个动物或器官成为一个想象设备，定量组织组织学要求每个组织切片，固定在载玻片上，扫描，单独分析。测试剂的标准制备并在相同的厚度器官样品切片。通过切割所有器官和标准，以相同的厚度，由于光散射或吸收可变性被消除，和组织的荧光强度可以配合到标准曲线来确定绝对concent配给。虽然这种方法中，当正确完成，是定量的，它也是劳动密集的，容易处理不当。给出定量组织学和劳动的显著成本较高的更复杂的性质相比整个器官成像时，它成为值得研究，其中每个过程是最实际的检验荧光标记的化合物的生物分布时使用。这个协议提供了如何这些方法可以一起使用，以有效地比较罗丹明标记的弹性蛋白样多肽（ELP）的生物分布，有或没有加入SYNB1或达细胞穿透肽的详细描述，通过鼻内（IN）和静脉内（IV）给药途径。

<table><tbody><tr><td>&lt;u&gt;Reagents&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Maleimide derivitized fluorophors (&lt;em&gt;e.g.&lt;/em&gt;, tetramethylrhodamine-5-maleimide, AlexaFluor 633-C5-maleimide)</td><td>Thermo Fisher</td><td>T6027, A20342</td><td>Thiol reactive fluorescent dyes for protein labeling</td></tr><tr><td>Phosphate Buffered Saline</td><td>Sigma</td><td>1002243569</td><td>PBS Buffer for rinsing</td></tr><tr><td>Optimal Cutting Temperature Compound</td><td>Tissue-Tek</td><td>4585</td><td>Used for freezing and mounting</td></tr><tr><td>&lt;u&gt;Equipment&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>IVIS Spectrum</td><td>Perkin Elmer</td><td></td><td>For &lt;em&gt;ex vivo&lt;/em&gt; whole organ imaging</td></tr><tr><td>Cryomicrotome</td><td>Thermo</td><td></td><td>For cryosectioning</td></tr><tr><td>Fluorescence slide scanner</td><td>Perkin Elmer</td><td></td><td>For slide scanning</td></tr></tbody></table>

the use of ex vivo whole-organ imaging and quantitative tissue histology to determine the bio-distribution of fluorescently labeled molecules

荧光标记是用于在各种体外和体内实验条件下检验标记的分子的命运成熟过程。荧光探针在确定施用大分子的生物分布，其中，除了一小分子荧光标记的是不太可能影响的动力学或化合物的生物分布是特别有用的。多种方法可用来检验生物分布，在努力所需的显著变化，并且所得到的测量结果是否完全定量的，但是使用在结合多个方法可以提供用于分析生物分布的快速和有效的系统。 离体全器官成像是可以用于对组织内和多种类型的组织或治疗组之间快速比较荧光分子的相对浓度的方法。使用成像开发平台无需进一步处理决定的形式完整的组织内专为活的动物或整个器官成像，荧光，省时省力，同时提供全面的生物分布的准确描述。这个过程是在实验试图确定的化合物的组织特异性或多个不同的化合物的比较理想的。另一方面定量组织组织学要求，以创建标记的化合物的定量测量组织的广泛进一步处理。准确评估生物分布，所有感兴趣的组织必须被切片，扫描，以使组织或组之间的比较相对于标准曲线进行分析。定量组织病理是确定组织中的绝对化合物浓度的黄金标准。 在这里，我们描述了如何两种方法可以一起有效地用于评估不同的给药方法的能力第二化合物修饰靶向和递送荧光标记的分子在中枢神经系统1。

下面的数据说明三种化合物的递送:被称为ELP和ELP的两个版本与细胞穿透肽（SYNB1-ELP和康达-ELP）9修饰的药物递送载体。所有这三个化合物标记四甲-5-马来酰亚胺和通过两个给药途径交付（IN和IV）。这些实验的目的是确定将导致最好渗透到中枢神经系统（CNS）3，其化合物和给药途径。 图1A显示代表离体从处理过的动物的器官的整个器官的图...

Watch this Scientific Journal Video about The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules at JoVE.com

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

离体全器官成像是用于确定内和组织或治疗组之间的荧光标记的化合物的相对浓度快速方法。相反，定量荧光组织学，而更多的劳动力密集的，允许对标记分子的绝对组织水平的量化。

指某东西的用途<em&gt;离体</em&gt;全器官成像和定量组织组织学确定荧光标记分子的生物分布

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

the-use-ex-vivo-whole-organ-imaging-quantitative-tissue-histology-to

Research

JoVE Journal

Biology

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

9.5K 次观看.  The University of Mississippi Medical Center.  离体全器官成像是用于确定内和组织或治疗组之间的荧光标记的化合物的相对浓度快速方法。相反，定量荧光组织学，而更多的劳动力密集的，允许对标记分子的绝对组织水平的量化。 

视频: The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

在肿瘤血管生成中使用荧光共聚焦电视显微镜活体成像

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

科研

医学

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 ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

混合μCT-FMT成像和图像分析

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

生物工程

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

使用宽场活细胞成像进行批量和单类器官分析的多参数肿瘤类器官药物筛选

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

癌症研究

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

体外肺转移及微环境的实时成像

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

应用先进的背皮窗室对转基因荧光小鼠肿瘤相关血管的活体显微镜观察

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

通过荧光报告基因定量进行全器官病原体追踪

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

作者聚焦:揭示细菌对组织中抗生素和免疫系统的反应

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

微生物感染的高分辨率三维全器官断层扫描

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

指某东西的用途

摘要

探索更多视频

此视频中的章节

在肿瘤血管生成中使用荧光共聚焦电视显微镜活体成像

一种新型的高分辨率

混合μCT-FMT成像和图像分析

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

使用宽场活细胞成像进行批量和单类器官分析的多参数肿瘤类器官药物筛选

体外肺转移及微环境的实时成像

应用先进的背皮窗室对转基因荧光小鼠肿瘤相关血管的活体显微镜观察

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

微生物感染的高分辨率三维全器官断层扫描

微生物感染的高分辨率三维全器官断层扫描