title: PhotoGate microscopy to track single molecules in crowded environments.
abstract: Tracking single molecules inside cells reveals the dynamics of biological processes, including receptor trafficking, signalling and cargo transport. However, individual molecules often cannot be resolved inside cells due to their high density. Here we develop the PhotoGate technique that controls the number of fluorescent particles in a region of interest by repeatedly photobleaching its boundary. PhotoGate bypasses the requirement of photoactivation to track single particles at surface densities two orders of magnitude greater than the single-molecule detection limit. Using this method, we observe ligand-induced dimerization of a receptor tyrosine kinase at the cell surface and directly measure binding and dissociation of signalling molecules from early endosomes in a dense cytoplasm with single-molecule resolution. We additionally develop a numerical simulation suite for rapid quantitative optimization of Photogate experimental conditions. PhotoGate yields longer tracking times and more accurate measurements of complex stoichiometry than existing single-molecule imaging methods.
authors.affiliation: Biophysics Graduate Group, University of California, Berkeley, California 94720, USA.
authors.forename: Vladislav
authors.initials: V
authors.lastname: Belyy
authors.affiliation: Department of Physics, University of California, Berkeley, California 94720, USA.
authors.forename: Sheng-Min
authors.initials: SM
authors.lastname: Shih
authors.affiliation: Department of Physics, University of California, Berkeley, California 94720, USA.
authors.forename: Jigar
authors.initials: J
authors.lastname: Bandaria
authors.affiliation: Biophysics Graduate Group, University of California, Berkeley, California 94720, USA.|Department of Molecular Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Yongjian
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Molecular Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Rosalie E
authors.initials: RE
authors.lastname: Lawrence
authors.affiliation: Department of Molecular Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Roberto
authors.initials: R
authors.lastname: Zoncu
authors.affiliation: Biophysics Graduate Group, University of California, Berkeley, California 94720, USA.|Department of Physics, University of California, Berkeley, California 94720, USA.|Department of Molecular Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Ahmet
authors.initials: A
authors.lastname: Yildiz
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D011992:Endosomes / Q000378:metabolism; D066246:ErbB Receptors / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D007834:Lasers; D008856:Microscopy, Fluorescence / Q000379:methods*; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods*; D057054:Molecular Imaging / Q000295:instrumentation / Q000379:methods*; D038761:Photobleaching; D055503:Protein Multimerization
chemical_list: C464801:APPL1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D049452:Green Fluorescent Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors