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使用量子点对多种内源性蛋白质进行相关光镜和电镜成像

Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots.

作者信息

Giepmans Ben N G, Deerinck Thomas J, Smarr Benjamin L, Jones Ying Z, Ellisman Mark H

机构信息

Department of Neurosciences, National Center for Microscopy and Imaging Research, and Center for Research in Biological Systems, University of California, San Diego, La Jolla, California, 92093-0608, USA.

出版信息

Nat Methods. 2005 Oct;2(10):743-9. doi: 10.1038/nmeth791.

Abstract

The importance of locating proteins in their context within cells has been heightened recently by the accomplishments in molecular structure and systems biology. Although light microscopy (LM) has been extensively used for mapping protein localization, many studies require the additional resolution of the electron microscope. Here we report the application of small nanocrystals (Quantum dots; QDs) to specifically and efficiently label multiple distinct endogenous proteins. QDs are both fluorescent and electron dense, facilitating their use for correlated microscopic analysis. Furthermore, QDs can be discriminated optically by their emission wavelength and physically by size, making them invaluable for multilabeling analysis. We developed pre-embedding labeling criteria using QDs that allows optimization at the light level, before continuing with electron microscopy (EM). We provide examples of double and triple immunolabeling using light, electron and correlated microscopy in rat cells and mouse tissue. We conclude that QDs aid precise high-throughput determination of protein distribution.

摘要

近期,分子结构和系统生物学领域所取得的成果进一步凸显了在细胞环境中定位蛋白质的重要性。尽管光学显微镜(LM)已被广泛用于绘制蛋白质定位图,但许多研究仍需要电子显微镜提供的更高分辨率。在此,我们报告了小纳米晶体(量子点;QDs)在特异性高效标记多种不同内源性蛋白质方面的应用。量子点既具有荧光性又具有电子致密性,便于用于相关显微镜分析。此外,量子点可通过发射波长进行光学区分,通过大小进行物理区分,这使其在多标记分析中具有极高价值。我们利用量子点制定了包埋前标记标准,该标准允许在进行电子显微镜(EM)观察之前,先在光学层面进行优化。我们提供了在大鼠细胞和小鼠组织中使用光学、电子和相关显微镜进行双重和三重免疫标记的示例。我们得出结论,量子点有助于精确高通量测定蛋白质分布。

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