Name: correlative light- and electron microscopy using quantum dot nanoparticles
Uploaded: 2016-08-07T14:00:00.000+00:00
Duration: 11 min 16 s
Description: Watch this Scientific Journal Video about Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles at JoVE.com

The authors wish to acknowledge the support of Xiao Juan Wu (Immunohistochemistry Laboratory) and the Department of Anatomical Pathology, Sydney South West Pathology Service (SSWPS), NSW Health Pathology, Liverpool, New South Wales, Australia.

1.组织剥离和固定<ol><li> 组织剥离 <ol><li>从手术切除肿瘤样品或组织活检解剖组织片。 注意:在此研究中使用的组织进行常规福尔马林固定，但新鲜组织也是合适的。我们选择了确认，并报告通过解剖病理学家以包含常规的组织学染色和抗促生长素抑制素的免疫染色（未示出）之后生长抑素瘤的区域。 </li><li>使用组织片不高于约1.0 立方毫米大。 </li></ol></li><li> 二甲胂酸缓冲液配制 <ol><li>通过加入21.4克二甲胂酸钠中制备储备缓冲溶液（1M）（见材料列表）和3.0毫升的1％氯化钙溶液至90毫升蒸馏水中，然后补足溶液至100ml的最终体积。搅拌溶液，并把它放置过夜使结晶完全试剂溶解。 </li><lI&gt;通过加入20毫升原液到170毫升水中，然后检查pH值试纸准备一个缓冲工作溶液。 3％的HCl 4滴 - 通过加入3将pH调节至7.4。补足溶液至200毫升的最终体积为0.1米的最终摩尔浓度</li></ol></li><li> 固色剂的制备和使用 <ol><li>的50％戊二醛溶液1个10 ml安瓿加入到20ml的二甲胂酸钠储备溶液（1 M）。用蒸馏水补足到190毫升检查pH值，并通过添加1调整到7.4 - 3％的盐酸（HCl）如必要4滴。 </li><li>补足蒸馏水，使200毫升的最终体积。 </li><li>浸入所述组织中含有2.5％戊二醛的0.1M二甲胂酸钠缓冲液pH 7.4，在4℃下，在一个玻璃样品管24小时的固定剂。 1周后丢弃未使用的固定液。 </li></ol></li></ol> 2.组织处理和嵌入<ol><li> 锇Tetro喜德染色<ol><li>固定完成后，浸泡在二甲胂酸钠缓冲液（0.1M）的组织20分钟，然后重复进行20分钟。 </li><li>通过加入8.0 5％OSO 4储备溶液的毫升制备的2％水溶液的四氧化锇（OSO 4）工作溶液到2.0毫升的二甲胂酸钠缓冲储备溶液（1μM）和10.0毫升的蒸馏水，使最终体积20.0毫升。 </li><li>除去则缓冲器浸入所述组织在2％OSO 4在pH 7.4在室温下4小时。 小心！调迁和准备OSO 4解决方案时要小心，穿适当的个人防护设备和工作在任何时候通风橱。 </li></ol></li><li> 醋酸铀染色 <ol><li>组织osmication完成后，取出OSO 4溶液并浸泡在2％的醋酸钠的组织10分钟。除去然后用2％醋酸钠在2％乙酸双氧铀浸泡在1小时室内温度。 </li></ol></li><li> 脱水 <ol><li>通过分级醇脱水组织。使用10分钟的50％乙醇，70％乙醇10分钟，95％乙醇10分钟，并在室温下每次20分钟，100％乙醇的两种变化。 </li><li>要对每个30分钟100％丙酮的两个变化。 </li></ol></li><li> 浸渍树脂和固化 <ol><li>在一次性聚乙烯杯中混合低粘度环氧树脂。添加10.0克乙烯基环己烯二氧化物（ERL 4221）环氧单体，6克聚丙二醇的二缩水甘油醚（DER 732）和26.0克壬烯基琥珀酸酐（NSA）的。用木棍2分钟用手充分搅拌。 </li><li>加入2二甲氨基乙醇（DMAE）环氧加速器十五滴2分钟再次搅拌。照顾到ERL，DER和NSA组件添加DMAE加速器前要充分混合。 </li><li> 1树脂:通过用1代替100％丙酮启动组织的树脂浸渍丙酮1小时，然后用6取代:1树脂丙酮3小时，最后100％树脂过夜。 </li><li>转移组织到新鲜树脂8毫米micromoulds。固化在70℃过夜。树脂要硬，但固化后不脆。 </li></ol></li></ol> 3.超薄切片<ol><li> 刀，切削参数和截面厚度 <ol><li>放置一个半薄金刚石刀在超薄切片机，在厚度为500nm的切割从组织切片。 </li><li>浮动部分到刀刃背后的水洗澡。拿起部分在载玻片上，并使用加热板，在约110℃。干燥下来进行10至20秒</li><li>蚀刻用乙醇钠溶液的部分20秒，然后用100％乙醇再用蒸馏水洗净。染色用2％的亚甲基中的1％硼砂蓝色持续20秒的热板，冲洗用流动的自来水进行5秒和干上热板5秒。然后STAIñ上的烤盘，持续5秒，然后在烤盘20秒干燥的1.5％碱性品红。 </li><li>由亮场光学显微镜检查，并确认肿瘤细胞和利息率（ROI）的区域中存在的部分。 </li><li>在半薄钻石刀改为超薄钻石刀并设置正确的刀角和切削速度所推荐的刀制造商。 </li><li> 90纳米厚切超薄切片。观察水浴漂浮时，各部分的黄金色。 </li><li>伸展并通过从区段的几毫米内1厘米2滤纸的挥动氯仿蒸气压平部分。小心不要把氯仿纸浸透与水的表面接触。 </li></ol></li><li> 超薄切片电网安装 <ol><li>从使用300目的细杆镍TEM网格已经在节adhesiv被浸入水表面抬起超薄切片e系统解决方案。找到对电网的沉闷侧部分。该网格的一侧和另一侧的抛光或光亮表面无光泽或亚光面。不支持膜是必需的。注意:有必要处理这些网格用抗磁镊子，以避免栅格粘到钳子。 </li><li>通过将20厘米透明胶纸在5毫升丙酮2.0毫升小瓶准备部分粘合。盖上盖子并晃动药瓶，静置10分钟，然后取出磁带离开粘合剂溶液。 </li></ol></li></ol> 4.免疫标记<ol><li> 抗原修复 <ol><li>制备将1ml蒸馏水中新鲜饱和偏高碘酸钠的蚀刻溶液，并立即使用它。在干净labfilm表面蚀刻溶液的地方液滴。 </li><li>放置完全干燥网格与部分向下的偏高碘酸钠溶液滴在室温下30分钟。转移网格来蒸馏水DROplets洗涤60秒。 </li></ol></li><li> 抗体孵育和QD探针标记 <ol><li>执行上放在干净的封口膜表面的飞沫解决所有的孵化和标签。 </li><li>上放置的0.05M的甘氨酸在PBS液滴电网10分钟，以阻断在部分残留的醛。通过短暂印迹网格的边缘除去醛块。 </li><li>放置在网格上的1％正常山羊血清（NGS）和1％BSAC（10％）的PBS 10分钟的液滴。通过加入NGS的10微升和100微升BSAC的890微升PBS中，得到1000微升的最终体积制备封闭溶液。 </li><li>通过在抗体稀释剂的液滴放置10分钟调节部分。 </li><li>执行使用与抗体稀释得到3.47克/升的浓度以1:10稀释的抗促生长素抑制素多克隆抗体的免疫标记孵育。注:抗体孵育在室温TEMPERAT放置网格上液滴1小时进行URE在潮湿小室。 </li><li>通过印迹网格的边缘去除步骤4.2.5抗体试剂然后洗部分上的稀释剂2×5分钟。 </li><li>孵育在室温下1小时，以1:10稀释的第二抗体（生物素化的山羊抗兔多克隆）。去除二级抗体溶液。洗稀释剂2×5分钟。 </li><li>孵育在室温下1小时，以1:10稀释的链霉轭量子点（585纳米）。用铝箔覆盖孵化过程中减少曝光。 </li><li>洗网格在新鲜蒸馏水2分钟。吸干网格干的边缘。 </li></ol></li></ol> 5.荧光光学显微镜<ol><li> 光源和滤镜立方体 <ol><li>使用365nm的发光二极管（LED），用于宽视场荧光光学显微镜的照明。插入一个过滤器立方体以下特点:（例摹365纳米，BS FT 395和EM LP 420纳米）。 注意:此过滤器立方W¯¯生病允许所有量子点的发射光谱尺寸要相同激发波长下同时可视化。 </li></ol></li><li> 查看和成像部件 <ol><li>放置免疫染色栅极部分，将其放在载玻片中的水滴，用玻璃盖玻片（厚度数1.5）覆盖。使用光学显微镜，以评估标记图案，任何非特异性标记的水平和以建立负控制清晰标记。 </li><li>确定投资回报率相对于电网的酒吧。取景网格可以是用于建立目标结构的坐标很有帮助。 </li><li>使用全彩色数码相机与"FL自动色彩"和"白平衡"设置为3,200K摄像机设置一节获得正面标签图像。使用"自动曝光"模式相机具有0.45和1.00之间的"伽玛"设置来优化图像的外观和"模拟增益"设定为1倍。点击禅2精简版软件的图形界面摄像头图标，"活"，注重图片，然后点击"捕捉"可将图像捕捉到的Framestore。 </li><li>图像保存为.TIF文件，以避免压缩和像素化开始显现。 </li><li>制备表示在ROI相对于网格条或其他显著组织的地标位置的打印。这些印刷品将是部分后续导航和重新发现通过透射电子显微镜（TEM）的ROI是有用的。 </li><li>从滑动取出网格，在蒸馏水中洗涤，并轻轻吸干边缘干燥。 注:请不要使用过于强烈的照明来获取荧光图像。量子点的光稳定性允许成像时间长达几秒钟，但要注意不要延长曝光的QD信号淬火后可在水中大约1分钟发生。当QD标记段是脱水甲苯只获得延长或永久的耐光性和嵌入式盖玻片下按照厂家的规范。然而，这个过程会再排除TEM同款的相关检查的可能性。 </li></ol></li></ol> 6.透射电子显微镜<ol><li> 透射电镜设置和查看 <ol><li>使用100千伏的加速电压转移网格在TEM检查。使用约1,400X的低倍率导航网四周，发现与光学显微镜相关意见投资回报。在约50,000X放大倍率量子点查看集群。 </li><li>请遵守70,000X放大或以上个人量子点。观察量子点为中度电子密度不规则的晶体结构。 </li></ol></li><li> 成像 <ol><li>使用TEM成像的数码相机。单色1392点¯x1,040像素传感器是足够的。 </li><li>通过点击"相机"图标为"开"的MICR获取图像oscope需要的成像软件的图形化界面，再到"关"的图像存储在帧存储。注意:这些操作将视显微镜系统上正在使用的不同。使用自动曝光模式下，相机为1.00一"伽玛"设置。 </li><li>图像保存为.TIF文件。 </li></ol></li></ol> 7. CLEM成像<ol><li> 通过查找一个TEM荧光投资回报率 <ol><li>从光镜图像用的打印定位由TEM相应的投资回报。组织建筑特色如血管和管腔结构是围绕用TEM的截面导航有用的。 </li><li>捕获通过TEM对应的ROI的图像。 </li></ol></li><li> CLEM图像覆盖 <ol><li>确保TEM图像被正确地与光学显微镜图像定向和校正每个的扩大，使得在每一个模态看到了同样的结构是相同的大小。 </li><li>东方吃了光镜和透射电镜照片，并显示他们并排侧或叠加凸显免疫标记感兴趣区域的超微结构特征。 </li><li>创建使用Photoshop CS2叠加图像。首先，放大荧光图像相同的放大倍数作为低功率TEM图像。定向两个图像，然后并排粘贴一边在一个画布。 </li><li>拼合图像。 </li><li>创建从该合成图像的荧光叠加。点击"矩形选框工具"，选择荧光图像，并从它创建一个副本层。 </li><li>调整"图层样式/混合选项"，以30 - 40％的透明度。 </li><li>点击"移动工具"和荧光覆盖层上相应的TEM图像拖动。对齐图像。 </li><li>实现对准之后，"拼合"层以产生最终叠加图像。 </li><li>使用"矩形选框工具"，选择新的叠加图像次将其复制到一个新的画布。保存为.tiff文件。 </li></ol></li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemistry+on+ultrathin+frozen+sections.">Immunochemistry on ultrathin frozen sections.</a> Histochem J. 12 (4), 381-403 (1980).</li><li>Loussert Fonta, C., 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=Analysis+of+acute+brain+slices+by+electron+microscopy:+a+correlative+light-electron+microscopy+workflow+based+on+Tokuyasu+cryo-sectioning.">Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.</a> J Struct Biol. 189 (1), 53-61 (2015).</li></ol>

The authors have no conflict of interest to disclose.

这项研究表明量子点的潜在效用为CLEM研究通用探头。由宽视场光学显微镜观察和透射电镜容易观察时使用的585纳米的纳米QD显示明亮，稳定的荧光。先前的研究由本作者之一显示量子点还适合于超分辨率光镜7。他们的耐光性是延长时间观看和长成像曝光特别有用。量子点也可以被用于以相同的激发波长下不同尺寸的探头的同时发射不同颜色的光谱多路复用免疫组织化学。 量子点具有足够的原子量，以通过TEM进行可视化，但是这被发现根据所用的纳米颗粒的大小要挑战。光镜，我们已经发现525纳米的量子点（绿色）相比较大的585纳米（橙色）和655纳米（红）的形式产生较少背景标记。然而，我们选择使用较大的585纳米的QD在本研究中，以使通过TEM标记更方便可视化。较小的525纳米的量子点的尺寸大约为3 - 5纳米，而较大的585纳米的形式是约6 - 8纳米和655纳米的量子点约8 - 10纳米。透射电镜观察时，585纳米量子点具有不规则形状的结晶中度电子密度。他们不是圆形或电子致密更传统的胶体金免疫细胞化学探针。然而，量子点已经显示出产量可达10倍更有效的标记比胶体金9和我们在这里确认了高标记密度是容易实现的，特别是与生物素-链霉探针联体系。的CdSe量子点还具有的特征元素的签名可以利用能量色散谱（EDS），电子能量损失谱（EELS）或能量过滤透射电镜检测和测绘允许（EFTEM）8。扫描透射电子显微镜（STEM）的系统也可由于其开发中的Z-对比度或原子序数成像这将加强纳米颗粒和低原子序数14的生物材料之间的对比使用。大面积的映射和与场发射扫描电子显微镜的三维分布信息15的可视化也应该是可以的。 在我们的方法中关键的一步是抗原修复或部分蚀刻过程。我们使用偏高碘酸钠的单一治疗在室温下30分钟。比这和结构或膜定义的损失的聚合更长的可能发生。他人已经使用涉及与甲醇钠，随后通过去osmication用高碘酸钠deplasticizing的两步方法，并在必要16，这可能提供更多的控制的过程。 CLEM是通过使用光镜小心ý登录有关尘棒的ROI的结构特征。然后同一网格放置在TEM和取向，使得尘棒和地标对应于光学显微镜图。大约1,400X的低倍率是最适合的。组织结构，如导管地层和原子核的图案被证明是重新找到的ROI的组织最有用的。然而，同一个小区的分辨率精确的相关性是很有挑战性的。护理不得不与电网的取向被视为反映通过光学显微镜看到的相同的方向。我们已经实现了与使用Photoshop制作叠加图像模式之间相当准确的亚细胞空间相关性。荧光叠加图像与TEM背景图像混合，但有些错位了检测。这可能是由于由每个模态或向我们的TEM不受支持超薄切片辐射损害所使用的不同的成像系统。对于ST自动化系统ORING光镜看到ROI的坐标和其传输至电子显微镜，现在正在销售，并会大大简化此过程。 我们已经提出了免疫细胞化学研究CLEM方法的效用是必然通过使用， 例如，重金属染色和环氧树脂包埋不可避免掩蔽可用的表位的样品制备方法的限制。然而，这项技术确实允许从病理样品获得荧光和TEM数据的可观的效益。获取使用单个样品从不同的显微镜模式的结构和功能信息是什么，也没有一般都提供给了处理，主要用于诊断目的，人的病理细胞和组织的研究。 它可以预期，更敏感的免疫细胞的定位可以从冷冻的固定方法，而不是一个协议B来获得ASED化学固定。冷冻固定的原则采用17对人体组织后cryoultramicrotomy和常温基于QD-免疫标记作为经典得安技术18生物样本库，19会促进人力发病机制的敏感性和特异性免疫相关性的研究。

相关轻型和电子显微镜（CLEM）是一种瞬态动态事件1，罕见的事件2,3和复杂系统4的分析功能强大的方法。有可用5取决于问题的许多不同技术的排列被要求然而一个共同要求是一个单一的样品6中相同的结构由多个显微镜模式成像。我们对特殊CLEM方法被用于存档人体病理学组织的研究，用在这里得到了很好的表征，此前公布的7的情况下开发的。其目的是首先从单个活检或手术样品其次最大化的分析数据，以使用荧光光学显微镜帮助阐明在超微结构水平看到的免疫细胞化学标记图案的情况下。 量子点纳米晶体（量子点）提供了一个通用的标记系统ABL的潜力由两个被看作即，荧光光镜和电镜8，9，10，它们的结晶核结构允许不同尺寸的量子点，当通过光波长远离其发射光谱11激发而产生的广泛的荧光发射峰。其原子量是足以产生电子密度是通过透射电子显微镜，扫描透射电子显微镜（STEM）或场发射扫描电子显微镜检测。它们特别适合于免疫细胞化学研究，作为甚至单个量子点可观察到给予每靶分子12 1 QD的最终的灵敏度。此外，根据不同的QD使用它们可具有适于映射的个体元素的签名。 人体病理学样品提供翻译生物医学研究显著的好处。外科组织活检标本例行提交的生物样本库和appropriate道德间隙可以为研究报告进行访问。人体组织不具有可发生在动物或在疾病的体外模型相关性或解释的问题。然而，病理学样品的样品制备通常不是最佳的。可存在于组织中的延迟被放置在固定剂，不适当的固定剂使用诸如福尔马林而非戊二醛用于TEM和不适当的采样。 CLEM方法必须优化可用从单个人类样本的诊断和预后信息的可能性。然而，一些新开发的相关的方法，例如那些采用微型单线态氧发生器（miniSOG）不适用于在病理学使用由于需要对标签被遗传编码到感兴趣13的细胞。为此，我们探索了常规准备TEM组织进行相关的免疫细胞化学研究的QD标签的效用。量子点应用于蚀刻环氧树脂或从李丙烯酸树脂切片ghtly醛固定活检组织样品提供从单个样品获得相关荧光光学显微镜和TEM数据的可能性。

<table><tbody><tr><td>Sodium cacodylate</td><td>Proscitech</td><td>C0205</td><td>Harmful chemical</td></tr><tr><td>Osmium tetroxide</td><td>Proscitech</td><td>C010</td><td>Use only in fume hood</td></tr><tr><td>Uranyl acetate</td><td>Univar-Ajax</td><td>569</td><td>Hazardous chemical</td></tr><tr><td>Ethanol 100%</td><td>Fronine</td><td>JJ008</td><td></td></tr><tr><td>Acetone 100%</td><td>Fronine</td><td>JJ006</td><td></td></tr><tr><td>ERL 4221</td><td>Proscitech</td><td>C056</td><td></td></tr><tr><td>DER 732</td><td>Proscitech</td><td>C047</td><td></td></tr><tr><td>NSA</td><td>Proscitech</td><td>C059</td><td></td></tr><tr><td>DMAE</td><td>Proscitech</td><td>C050</td><td></td></tr><tr><td>Sodium metaperiodate</td><td>Analar BDH</td><td>10259</td><td></td></tr><tr><td>anti-somatostatin antibody</td><td>Dako</td><td>A0566</td><td></td></tr><tr><td>Antibody diluent</td><td>Dako</td><td>S3022</td><td></td></tr><tr><td>Qdot 585 Streptavidin Conjugate&amp;nbsp;</td><td>Invitrogen</td><td>Q10113MP</td><td></td></tr><tr><td>Biotinylated goat anti-rabbit IgG antibody</td><td>Sigma</td><td>B7389-1ML</td><td></td></tr><tr><td>Glutaraldehyde 50%</td><td>EMS</td><td>16320</td><td></td></tr><tr><td>Normal goat serum</td><td>Invitrogen</td><td>PCN5000</td><td></td></tr><tr><td>PBS &quot;Dulbecco A&quot;</td><td>Oxoid&amp;nbsp;</td><td>BR0014G</td><td></td></tr><tr><td>BSAc (10%)</td><td>Aurion</td><td>900.022</td><td></td></tr><tr><td>Parafilm</td><td>Pechiney PP</td><td>M</td><td></td></tr><tr><td>pH indicator strips (pH 2.0 - 9.0)</td><td>Merck</td><td>1.09584.0001</td><td></td></tr><tr><td>Micromoulds</td><td>Proscitech</td><td>RL063</td><td></td></tr><tr><td>Diamond knife</td><td>Diatome</td><td>Ultra 45</td><td></td></tr><tr><td>Transmission electron microscope</td><td>FEI</td><td>Morgagni 268D</td><td></td></tr><tr><td>Fluorescence light microscope</td><td>Carl Zeiss</td><td>Axioscope A1</td><td></td></tr><tr><td>Grids 300 mesh nickel (thin bar)</td><td>Agar Scientific</td><td>G2740N</td><td></td></tr><tr><td>Ultramicrotome</td><td>RMC</td><td>Powertome</td><td></td></tr><tr><td>TEM camera control software</td><td>Soft Imaging System</td><td>AnalySIS</td><td>Version 3.0</td></tr><tr><td>Image processing software</td><td>Adobe Systems Incorporated</td><td>Photoshop CS2</td><td></td></tr></tbody></table>

correlative light- and electron microscopy using quantum dot nanoparticles

描述了一种方法，即量子点（QD）纳米颗粒可用于使用宽视场荧光光镜和透射电子显微镜（TEM）人体病理学组织相关的免疫细胞化学研究。为了证明我们已经用初级抗体对促生长素抑制素免疫标记人生长抑素瘤的超薄环氧部分的协议，其次是生物素化的二级抗体和可视化用链霉缀585nm的镉 - 硒（硒化镉）量子点（QDs）。的部分被安装在TEM样品网格，然后通过宽视场荧光光镜放置在载玻片观察。光镜显示585纳米量子点标记为明亮的橙色荧光形成肿瘤细胞的细胞质中的颗粒图案。在通过光学显微镜低中档放大率标签图案可以容易地识别和非特异性或背景标记的水平进行评估。这是一个关键步骤，通过TEM免疫标记图案的后续解释和评价形态上下文。然后相同部分被吸干并用TEM观察。 QD探针被视为要附加到包含在个别分泌颗粒无定形物质。图像是从减光镜进行相关性分析可见感兴趣区域（ROI）的同一区域收购。从每个模态对应的图像随后可以被混合以覆盖上相应的区域的TEM超微结构荧光数据。

用于该研究的生长抑素瘤样品包括形成具有胶原造口组织混合导管结构的肿瘤细胞。通过荧光光镜，含有丰富的分泌颗粒个体肿瘤细胞表现为生长抑素激素阳性标记。细胞核出现暗孔以最小非特异性标记检测的（ 图1）。在低倍率，可变地激烈粒状橙色荧光被认为在这些充分表征的肿瘤细胞的细胞质中。荧光的橙色由QD使用的大小决定。在这项研究中，我们使用585纳米的量子点时365纳米的光激发时发出橙色信号。 同一小区的CLEM分析是可能的（ 图2）。使用相同的网格，通过光学显微镜透射电镜观察允许的投资回报看，以识别和IM在岁的两种模式。光镜所见有关尘棒相同的组织结构用TEM观察和用于导航。在ROI被通过参照一个20X光学显微镜图像，并注意到组织特性相对于尘棒找到。 TEM示生长抑素阳性细胞的细胞质中含有不同电子密度的丰富圆形颗粒。各种直径的颗粒存在于个体细胞，并通过颗粒QD免疫标记密度也是可变的。关于TEM图像的荧光数据的叠加表明强阳性标记对应于含有囊泡界膜（ 图3）内适度电子致密材料颗粒。 在高放大倍率的电子适度致密颗粒可以看到与QD纳米晶体（被强烈的免疫标记<stroNG&gt;图4）。 <img alt="图1" src="/files/ftp_upload/54307/54307fig1.jpg" /> 图1:一个超薄节与标记为生长抑素激素生长抑素细胞光学显微镜观的超薄切片安装在TEM网格，免疫标记，然后放置载玻片上在水中的盖玻片下。生长抑素细胞（箭头）显示在细胞质中圆形核和丰富生长抑素激素阳性颗粒。贴标密度相当大的变化是出现在生长抑素细胞群体（200X）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54307/54307fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54307/54307fig2.jpg" /> 图2:CLEM复合视图从超薄切片通过生长抑素<html> 。标记生长抑素一个 OMA组织），由宽视场荧光显微镜生长抑素细胞（120X）; B）围绕在低倍率（570X）通过TEM看到流明（L）同细胞; C）表示从明亮的荧光细胞高功率TEM视图（ A）与核（N）（2000倍）; D）细胞质颗粒显示超过所含颗粒（星号）（20000）中的非晶材料量子点标记激烈的详细信息<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54307/54307fig2large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54307/54307fig3.jpg" /> 图3:CLEM叠加图像从与相应区域（2000倍）的TEM图像混合荧光显微镜透明图像。/ftp_upload/54307/54307fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54307/54307fig4.jpg" /> 图 4: 从在细胞质中（箭头）显示QD定位在非晶内容很大程度上限制膜内生长抑素细胞质生长抑素激素阳性颗粒生长抑素阳性颗粒的透射电子显微镜（TEM）观 。周围细胞质背景是干净的很少QD探头明显。一些非特定的核标记被看到。周围的颗粒（箭头）的小泡膜仍可见（50,000X）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54307/54307fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles at JoVE.com

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

correlative-light-electron-microscopy-using-quantum-dot

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.6K 次观看.  University of New South Wales Australia. A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

视频: Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

紧凑型量子点的单分子成像

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

科研

工程学

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

低成本的低温光学显微镜舞台制作相关的光/电子显微镜

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

生物工程

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

低温固定化细胞跟踪和成像病毒蛋白相关结构的相关光电子显微镜 (克莱)

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

生物学

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

纳米有限元分析：蛋白质定位的光激活定位显微镜和电子显微镜

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Correlative Microscopy Reveals Stress-Induced Mitochondria-Lysosome Interactions

Dual-color Correlative Light and Electron Microscopy for the Visualization of Interactions between Mitochondria and Lysosomes

Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for structural analysis, light microscopy is essential for the visualization of dynamic organelles by target-specific labeling. The following protocol describes a method that combines dual-color correlative light and electron microscopy (CLEM) to observe the interactions between mitochondria and lysosomes. In this study, mitochondria were labeled with mEosEM (Mito-mEosEM) and lysosomes with TMEM192-V5-APEX2. The results obtained from CLEM images enable us to observe the changes in the interactions between mitochondria and lysosomes under external stress conditions. Treatment with bafilomycin (BFA), which inhibits lysosomal function, resulted in an increase in contact between mitochondria and lysosomes, leading to the formation of fragmented mitochondria trapped inside lysosomes. Conversely, treatment with U18666A, which inhibits cholesterol export from lysosomes, caused lysosomes to be surrounded by mitochondria, indicating a distinct form of interaction. This study presents an effective method for observing the interactions between mitochondria and lysosomes in fixed cells. Furthermore, CLEM imaging with dual-color probes offers a powerful tool for future investigations of organelle dynamics and their implications for cell function and pathology.

This protocol describes a mechanism for using correlative light and electron microscopy to visualize the interaction of mitochondria and lysosomes labeled with mEosEM and APEX2, respectively.

Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for ...

神经科学

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

In this protocol, quantum dots conjugated to recombinant SARS-CoV-2 spike enable cell-based assays to monitor spike binding to hACE2 at the plasma membrane and subsequent endocytosis of the bound proteins into the cytoplasm.

量子点偶联SARS-CoV-2穗状三聚体的高通量共聚焦成像，以追踪HEK293T细胞中的结合和内吞作用

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum ...

Visualizing Subcellular Localization of a Protein in the Heart Using Quantum Dots-Mediated Immuno-Labeling Followed by Transmission Electron Microscopy

The subcellular localization is critical to delineating proper function and determining the molecular mechanisms of a particular protein. Several qualitative and quantitative techniques are used to determine the subcellular localization of proteins. One of the emerging techniques in determining the subcellular localization of a protein is quantum dots (QD)-mediated immunolabeling of a protein followed by imaging them with transmission electron microscopy (TEM). QD is a semiconductor nanocrystal with a dual property of crystalline structure and high electron density, which makes them applicable to electron microscopy. This current method visualized the subcellular localization of Sigma 1 receptor (Sigmar1) protein using QD-TEM in the heart tissue at ultrastructural level. Small cubes of the heart tissue sections from a wild-type mouse were fixed in 3% glutaraldehyde, subsequently osmicated, stained with uranyl acetate, followed by sequential dehydration with ethanol and acetone. These dehydrated heart tissue sections were embedded in low-viscosity epoxy resins, cut into thin sections of 500 nm thickness, put on the grid, and subsequently subjected to antigen unmasking with 5% sodium metaperiodate, followed by quenching of the residual aldehydes with glycine. The tissues were blocked, followed by sequential incubation in primary antibody, biotinylated secondary antibody, and streptavidin-conjugated QD. These stained sections were blot dried and imaged at high magnification using TEM. The QD-TEM technique allowed the visualization of Sigmar1 protein&#39;s&#160;subcellular localization at the ultrastructural level in the heart. These techniques can be used to visualize the presence of any protein and subcellular localization in any organ system.

The present protocol describes a method of immuno-labeling a protein in the heart tissue sections using quantum dots. This technique provides a useful tool to visualize any protein&#39;s subcellular localization and expression at the ultrastructural level.

使用量子点介导的免疫标记，然后通过透射电子显微镜可视化心脏中蛋白质的亚细胞定位

The subcellular localization is critical to delineating proper function and determining the molecular mechanisms of a particular protein. Several ...

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view.
In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification.
In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.

Here, we present a protocol for optimized on-section correlative light-electron microscopy based on endogenous, fluorescent labeling as a tool to investigate the localization of rare proteins in relation to cellular ultrastructure. The power of this approach is demonstrated by ultrastructural localization of endogenous LC3 in starved cells without Bafilomycin treatment.

内源性LC3的超微结构定位-截面相关光电子显微镜

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal ...

相关轻型和电子显微镜使用量子点纳米粒子

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