Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples

作者：Kopek Benjamin G; Paez Segala Maria G; Shtengel Gleb; Sochacki Kem A; Sun Mei G; Wang Yalin; Xu C Shan; van Engelenburg Schuyler B; Taraska Justin W; Looger Loren L; Hess Harald F

来源：Nature Protocols, 2017, 12(5): 916-946.

DOI：10.1038/nprot.2017.017

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 data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The 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 replication creates high-contrast, 3D 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 (similar to 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 d, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.

• 出版日期2017-5