title: Visualizing proteins in electron micrographs at nanometer resolution.
abstract: To understand protein function, we need a detailed description of the molecular topography of the cell. The subcellular localization of proteins can be revealed using genetically encoded fluorescent proteins or immunofluorescence. However, the precise localization of proteins cannot be resolved due to the diffraction limit of light. Recently, the diffraction barrier has been overcome by employing several microscopy techniques. Using super-resolution fluorescence microscopy, one can pinpoint the location of proteins at a resolution of 20 nm or even less. However, the cellular context is often absent in these images. Recently, we developed a method for visualizing the subcellular structures in super-resolution images. Here we describe the method with two technical improvements. First, we optimize the method to preserve more fluorescence without compromising the morphology. Second, we implement ground-state depletion and single-molecule return (GSDIM) imaging, which does not rely on photoactivatable fluorescent proteins. These improvements extend the utility of nano-resolution fluorescence electron microscopy (nano-fEM).
authors.affiliation: Howard Hughes Medical Institute and Department of Biology, University of Utah, Salt Lake City, UT 84112-0840, USA.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Watanabe
authors.forename: Erik M
authors.initials: EM
authors.lastname: Jorgensen
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D015925:Cryopreservation; D008164:Luminescent Proteins / Q000096:biosynthesis; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D008867:Microtomy; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D016613:Plastic Embedding; D011994:Recombinant Proteins / Q000096:biosynthesis; D016707:Tissue Fixation
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C434479:citrine protein, bacteria