title: Real-time fluorescence imaging with 20 nm axial resolution.
abstract: Measuring the nanoscale organization of protein structures near the plasma membrane of live cells is challenging, especially when the structure is dynamic. Here we present the development of a two-wavelength total internal reflection fluorescence method capable of real-time imaging of cellular structure height with nanometre resolution. The method employs a protein of interest tagged with two different fluorophores and imaged to obtain the ratio of emission in the two channels. We use this approach to visualize the nanoscale organization of microtubules and endocytosis of the epidermal growth factor receptor.
authors.affiliation: Department of Chemistry, Emory University, Atlanta, Georgia 30322, USA.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Stabley
authors.affiliation: Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Oh
authors.affiliation: Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Sanford M
authors.initials: SM
authors.lastname: Simon
authors.affiliation: Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: Alexa L
authors.initials: AL
authors.lastname: Mattheyses
authors.affiliation: Department of Chemistry, Emory University, Atlanta, Georgia 30322, USA.
authors.forename: Khalid
authors.initials: K
authors.lastname: Salaita
mesh_terms: D002462:Cell Membrane / Q000737:chemistry*; D005456:Fluorescent Dyes; D008870:Microtubules; D061848:Optical Imaging / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins