相关轻型和电子显微镜使用量子点纳米粒子
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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。
<p class="jove_co…Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Sodium cacodylate | Proscitech | C0205 | Harmful chemical |
| Osmium tetroxide | Proscitech | C010 | Use only in fume hood |
| Uranyl acetate | Univar-Ajax | 569 | Hazardous chemical |
| Ethanol 100% | Fronine | JJ008 | |
| Acetone 100% | Fronine | JJ006 | |
| ERL 4221 | Proscitech | C056 | |
| DER 732 | Proscitech | C047 | |
| NSA | Proscitech | C059 | |
| DMAE | Proscitech | C050 | |
| Sodium metaperiodate | Analar BDH | 10259 | |
| anti-somatostatin antibody | Dako | A0566 | |
| Antibody diluent | Dako | S3022 | |
| Qdot 585 Streptavidin Conjugate | Invitrogen | Q10113MP | |
| Biotinylated goat anti-rabbit IgG antibody | Sigma | B7389-1ML | |
| Glutaraldehyde 50% | EMS | 16320 | |
| Normal goat serum | Invitrogen | PCN5000 | |
| PBS "Dulbecco A" | Oxoid | BR0014G | |
| BSAc (10%) | Aurion | 900.022 | |
| Parafilm | Pechiney PP | M | |
| pH indicator strips (pH 2.0 – 9.0) | Merck | 1.09584.0001 | |
| Micromoulds | Proscitech | RL063 | |
| Diamond knife | Diatome | Ultra 45 | |
| Transmission electron microscope | FEI | Morgagni 268D | |
| Fluorescence light microscope | Carl Zeiss | Axioscope A1 | |
| Grids 300 mesh nickel (thin bar) | Agar Scientific | G2740N | |
| Ultramicrotome | RMC | Powertome | |
| TEM camera control software | Soft Imaging System | AnalySIS | Version 3.0 |
| Image processing software | Adobe Systems Incorporated | Photoshop CS2 |
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