title: Nanometric molecular separation measurements by single molecule photobleaching.
abstract: Although considerable progress has been made in imaging distances in cells below the diffraction limit using FRET and super-resolution microscopy, methods for determining the separation of macromolecules in the 10-50 nm range have been elusive. We have developed fluorophore localisation imaging with photobleaching (FLImP), based on the quantised bleaching of individual protein-bound dye molecules, to quantitate the molecular separations in oligomers and nanoscale clusters. We demonstrate the benefits of using our method in studying the nanometric organisation of the epidermal growth factor receptor in cells.
authors.affiliation: Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Campus, Didcot OX11 0FA, UK.
authors.forename: Stephen E D
authors.initials: SE
authors.lastname: Webb
authors.affiliation: Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Campus, Didcot OX11 0FA, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Hirsch
authors.affiliation: Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Campus, Didcot OX11 0FA, UK.
authors.forename: Sarah R
authors.initials: SR
authors.lastname: Needham
authors.affiliation: Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Campus, Didcot OX11 0FA, UK.
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Coles
authors.affiliation: Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Campus, Didcot OX11 0FA, UK.
authors.forename: Kathrin M
authors.initials: KM
authors.lastname: Scherer
authors.affiliation: Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Campus, Didcot OX11 0FA, UK.
authors.forename: Selene K
authors.initials: SK
authors.lastname: Roberts
authors.affiliation: Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Campus, Didcot OX11 0FA, UK.
authors.forename: Laura C
authors.initials: LC
authors.lastname: Zanetti-Domingues
authors.affiliation: Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Campus, Didcot OX11 0FA, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Tynan
authors.affiliation: Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Campus, Didcot OX11 0FA, UK.
authors.forename: Marisa L
authors.initials: ML
authors.lastname: Martin-Fernandez
authors.affiliation: Central Laser Facility, Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Harwell Campus, Didcot OX11 0FA, UK. Electronic address: daniel.rolfe@stfc.ac.uk.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Rolfe
mesh_terms: D000818:Animals; D006224:Cricetinae; D066246:ErbB Receptors / Q000737:chemistry*; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D038761:Photobleaching
chemical_list: D005456:Fluorescent Dyes; D046911:Macromolecular Substances; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: Dimerisation; Epidermal growth factor receptor; Oligomerisation; Single-molecule localisation