title: Stochastic approach to the molecular counting problem in superresolution microscopy.
abstract: Superresolution imaging methods--now widely used to characterize biological structures below the diffraction limit--are poised to reveal in quantitative detail the stoichiometry of protein complexes in living cells. In practice, the photophysical properties of the fluorophores used as tags in superresolution methods have posed a severe theoretical challenge toward achieving this goal. Here we develop a stochastic approach to enumerate fluorophores in a diffraction-limited area measured by superresolution microscopy. The method is a generalization of aggregated Markov methods developed in the ion channel literature for studying gating dynamics. We show that the method accurately and precisely enumerates fluorophores in simulated data while simultaneously determining the kinetic rates that govern the stochastic photophysics of the fluorophores to improve the prediction's accuracy. This stochastic method overcomes several critical limitations of temporal thresholding methods.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158;
authors.forename: Geoffrey C
authors.initials: GC
authors.lastname: Rollins
authors.affiliation: Howard Hughes Medical Institute and.
authors.forename: Jae Yen
authors.initials: JY
authors.lastname: Shin
authors.affiliation: Howard Hughes Medical Institute and Departments of Physics, Molecular and Cell Biology, and Chemistry, QB3 Institute, University of California, Berkeley, CA 94720;
authors.forename: Carlos
authors.initials: C
authors.lastname: Bustamante
authors.affiliation: Department of Physics, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202; and Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202 stevenpresse@gmail.com.
authors.forename: Steve
authors.initials: S
authors.lastname: Pressé
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry; D016013:Likelihood Functions; D046911:Macromolecular Substances / Q000737:chemistry*; D008390:Markov Chains; D008853:Microscopy / Q000379:methods* / Q000706:statistics & numerical data; D008856:Microscopy, Fluorescence / Q000379:methods / Q000706:statistics & numerical data; D008956:Models, Chemical; D057054:Molecular Imaging / Q000379:methods / Q000706:statistics & numerical data; D046912:Multiprotein Complexes / Q000737:chemistry; D055668:Photochemical Processes; D013269:Stochastic Processes
chemical_list: D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D046912:Multiprotein Complexes
keywords: counting problem; fluorescence; protein complexes; single molecule; superresolution