title: Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders.
abstract: With the recent development of single-molecule localization-based superresolution microscopy, the imaging of cellular structures at a resolution below the diffraction-limit of light has become a widespread technique. While single fluorescent molecules can be resolved in the nanometer range, the delivery of these molecules to the authentic structure in the cell via traditional antibody-mediated techniques can add substantial error due to the size of the antibodies. Accurate and quantitative labeling of cellular molecules has thus become one of the bottlenecks in the race for highest resolution of target structures. Here we illustrate in detail how to use small, high affinity nanobody binders against GFP and RFP family proteins for highly generic labeling of fusion constructs with bright organic dyes. We provide detailed protocols and examples for their application in superresolution imaging and single particle tracking and demonstrate advantages over conventional labeling approaches.
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, SE1 1UL, United Kingdom; Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.
authors.forename: Evgenia
authors.initials: E
authors.lastname: Platonova
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, SE1 1UL, United Kingdom; Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.
authors.forename: Christian M
authors.initials: CM
authors.lastname: Winterflood
authors.affiliation: Hannover Medical School, Hannover, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Junemann
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, SE1 1UL, United Kingdom; Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Albrecht
authors.affiliation: Hannover Medical School, Hannover, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Faix
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, SE1 1UL, United Kingdom; Institute of Biochemistry, ETH Zurich, Zurich, Switzerland; Institut für Biochemie und Chemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany. Electronic address: helge.ewers@fu-berlin.de.
authors.forename: Helge
authors.initials: H
authors.lastname: Ewers
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000276:immunology*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000276:immunology*; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods; D051381:Rats; D061905:Single-Domain Antibodies; D013194:Staining and Labeling / Q000379:methods*; D000097573:Red Fluorescent Protein
chemical_list: D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D061905:Single-Domain Antibodies; D000097573:Red Fluorescent Protein
keywords: Nanobody; PALM; STORM; Single domain antibody; Single particle tracking (SPT); Single-molecule localization microscopy (SMLM); Spectral-demixing direct stochastic reconstruction microscopy (SD-dSTORM); VHH