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      Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

      Author(s): , , , ,
      Publication date Created:
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      Journal: Nature Protocols
      Publisher: Springer Nature

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          Abstract

          This protocol describes procedures for using the genetic tag APEX2 to generate contrast for electron microscopy in cultured cells.

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          The fluorescent toolbox for assessing protein location and function.

          Ben Giepmans, Stephen R Adams, Mark Ellisman … (2006)
          Advances in molecular biology, organic chemistry, and materials science have recently created several new classes of fluorescent probes for imaging in cell biology. Here we review the characteristic benefits and limitations of fluorescent probes to study proteins. The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small organic fluorescent dyes, nanocrystals ("quantum dots"), autofluorescent proteins, small genetic encoded tags that can be complexed with fluorochromes, and combinations of these probes are highlighted.
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            Directed evolution of APEX2 for electron microscopy and proteomics

            Stephanie Lam, Jeffrey D. Martell, Kimberli J. Kamer … (2014)
            APEX is an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. Because the limited sensitivity of APEX precludes applications requiring low APEX expression, we used yeast display evolution to improve its catalytic efficiency. Our evolved APEX2 is far more active in cells, enabling the superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins and the use of electron microscopy to resolve the sub-mitochondrial localization of calcium uptake regulatory protein MICU1.
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              Fluorescent probes for super-resolution imaging in living cells.

              Marta Fernandez-Suarez, Alice Ting (2008)
              In 1873, Ernst Abbe discovered that features closer than approximately 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.
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                Author and article information

                Journal
                Title: Nature Protocols
                Abbreviated Title: Nat Protoc
                Publisher: Springer Nature
                ISSN (Print): 1754-2189
                ISSN (Electronic): 1750-2799
                Publication date Created: August 10 2017
                Publication date (Print): August 10 2017
                Volume: 12
                Issue: 9
                Pages: 1792-1816
                Article
                DOI: 10.1038/nprot.2017.065
                PMC ID: 5851282
                PubMed ID: 28796234
                SO-VID: 8d74d2e5-59bf-4f9e-bb70-cc7b10c6b996
                Copyright © © 2017
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