Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots
Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots
复制标题
DOI：
10.1038/nmeth791
复制
发表时间：
2005-10-01
期刊：
影响因子：
48
通讯作者：
Ellisman, MH
中科院分区：
文献类型：
--
作者：
Giepmans, BNG;Deerinck, TJ;Ellisman, MH
最近，分子结构和系统生物学的成就提高了在细胞内定位蛋白质的重要性。虽然紧密显微镜（LM）已被广泛用于映射蛋白质定位，许多研究需要额外的分辨率的电子显微镜。在这里，我们报告的应用程序的小纳米晶体（量子点;量子点）特异性和有效地标记多种不同的内源性蛋白质。量子点是荧光和电子致密的，便于它们用于相关的显微镜分析。此外，量子点可以通过它们的发射波长和物理大小进行光学区分，这使得它们对于多标记分析非常宝贵。我们开发了预包埋标记标准，使用量子点，允许优化的光水平，然后继续与电子显微镜（EM）。我们提供了在大鼠细胞和小鼠组织中使用紧密，电子和相关显微镜进行双重和三重免疫标记的例子。我们得出结论，量子点有助于精确的高通量测定蛋白质分布。
The importance of Locating proteins in their context within cells has been heightened recently by the accomplishments in molecular structure and systems biology. Although tight 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 tight, electron and correlated microscopy in rat cells and mouse tissue. We conclude that QDs aid precise high-throughput determination of protein distribution.