title: Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples.
abstract: 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 (∼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.
authors.affiliation: Department of Biology, Hope College, Holland, Michigan, USA.
authors.forename: Benjamin G
authors.identifier: 0000-0002-5288-3744
authors.initials: BG
authors.lastname: Kopek
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Maria G
authors.initials: MG
authors.lastname: Paez-Segala
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Gleb
authors.initials: G
authors.lastname: Shtengel
authors.affiliation: National Heart, Lung, and Blood Institute, US National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Kem A
authors.initials: KA
authors.lastname: Sochacki
authors.affiliation: Pathology Division, US Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA.
authors.forename: Mei G
authors.initials: MG
authors.lastname: Sun
authors.affiliation: Advanced Microscopy Facility, University of Virginia School of Medicine, Charlottesville, Virginia, USA.
authors.forename: Yalin
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: C Shan
authors.initials: CS
authors.lastname: Xu
authors.affiliation: Department of Biological Sciences, University of Denver, Denver, Colorado, USA.
authors.forename: Schuyler B
authors.initials: SB
authors.lastname: van Engelenburg
authors.affiliation: National Heart, Lung, and Blood Institute, US National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Justin W
authors.initials: JW
authors.lastname: Taraska
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Loren L
authors.initials: LL
authors.lastname: Looger
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Harald F
authors.initials: HF
authors.lastname: Hess
mesh_terms: D001688:Biological Products / Q000032:analysis*; D003584:Cytological Techniques / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*
chemical_list: D001688:Biological Products