title: A simple method for GFP- and RFP-based dual color single-molecule localization microscopy.
abstract: The recent development of single-molecule localization-based super-resolution techniques has afforded a resolution in the nanometer range in light microscopy. The ability to resolve biological structures on this scale by multicolor techniques faces significant challenges which have prevented their widespread use. Here, we provide a generic approach for high-quality simultaneous two-color single-molecule localization microscopy imaging of any combination of GFP- and RFP-tagged proteins with the use of nanobodies. Our method addresses a number of common issues related to two-color experiments, including accuracy and density of labeling as well as chromatic aberration and color-crosstalk with only minimal technical requirements. We demonstrate two-color imaging of various nanoscopic structures and show a compound resolution down to the limit routinely achieved only in a single color.
authors: †Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, United Kingdom.|§Institute of Biochemistry, ETH Zurich, Otto-Stern-Weg 3, 8093 Zurich, Switzerland.
authors: Evgenia
authors: E
authors: Platonova
authors: †Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, United Kingdom.|§Institute of Biochemistry, ETH Zurich, Otto-Stern-Weg 3, 8093 Zurich, Switzerland.
authors: Christian M
authors: CM
authors: Winterflood
authors: †Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, United Kingdom.|‡Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.|§Institute of Biochemistry, ETH Zurich, Otto-Stern-Weg 3, 8093 Zurich, Switzerland.
authors: Helge
authors: H
authors: Ewers
mesh_terms: D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D003116:Color; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D061905:Single-Domain Antibodies / Q000737:chemistry*; D000097573:Red Fluorescent Protein
chemical_list: D051242:Caveolin 1; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D028861:Nuclear Pore Complex Proteins; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D000097573:Red Fluorescent Protein; C495540:CAV1 protein, human; C079377:NUP153 protein, human; C485184:enhanced green fluorescent protein; C049312:nuclear pore protein p62