Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples.
Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples.
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DOI：
10.1038/nprot.2017.017
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发表时间：
2017-05
期刊：
影响因子：
14.8
通讯作者：
Hess HF
中科院分区：
文献类型：
--
作者：
Kopek BG;Paez-Segala MG;Shtengel G;Sochacki KA;Sun MG;Wang Y;Xu CS;van Engelenburg SB;Taraska JW;Looger LL;Hess HF
我们的团队最近开发了相关的方法，用于内源性细胞环境中使用相关光和电子显微镜（CLEM）进行超分辨率成像的样品制备。提供了四种不同的制备和获取超分辨率CLEM数据集的方法，包括Tokuyasu冷冻切片，全细胞贴载，细胞无顶和铂复制，以及树脂包埋和切片。为给定的应用程序选择最佳协议取决于我们将详细讨论的许多标准。德康冷冻切片相对快速，但仅限于小而精致的标本。全细胞贴装具有最简单的样品制备，但仅限于表面结构。细胞剥离和铂复制可以创建高对比度的质膜细胞质表面的三维图像，但比整个细胞安装更具挑战性。树脂包埋允许大样品的连续切片，但仅限于抗锇探针，在技术上是困难的。这些方案的预期结果包括在电子显微镜下超分辨率定位（~ 10-50 nm）荧光靶标，这可以帮助解决细胞生物学问题。这些方案可在2-7天内完成，与许多超分辨率成像方案兼容，并广泛适用于整个生物学。
Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM datasets on aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. Choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replica creates high-contrast, 3-dimensional images of the cytoplasmic surface of the plasma membrane, but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples, but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (~10–50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2–7 days, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.
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影响因子：
14.8
作者：
Gould, Travis J.;Verkhusha, Vladislav V.;Hess, Samuel T.
通讯作者：
Hess, Samuel T.
影响因子：
4.6
作者：
Johnson E;Seiradake E;Jones EY;Davis I;Grünewald K;Kaufmann R
通讯作者：
Kaufmann R
DOI：
10.1073/pnas.0907866106
发表时间：
2009-12-29
影响因子：
11.1
作者：
Dertinger, T.;Colyer, R.;Enderlein, J.
通讯作者：
Enderlein, J.
影响因子：
9.2
作者：
Collins, Agnieszka;Warrington, Anthony;Taylor, Kenneth A.;Svitkina, Tatyana
通讯作者：
Svitkina, Tatyana
影响因子：
10.8
作者：
Kaufmann R;Schellenberger P;Seiradake E;Dobbie IM;Jones EY;Davis I;Hagen C;Grünewald K
通讯作者：
Grünewald K