title: Intrinsically resolution enhancing probes for confocal microscopy.
abstract: In recent years different implementations of superresolution microscopy based on targeted switching (STED, GSD, and SSIM) have been demonstrated. The key elements to break the diffraction barrier are two distinct molecular states that generate a saturable nonlinear fluorescence response with respect to the excitation intensity. In this paper, we demonstrate that a nonlinearity can even be encoded in fluorescent probes, which then increase the resolution of a standard confocal microscope. This nonlinearity is achieved by an intensity dependent blocking of the resonance energy transfer between a donor and one or more acceptor fluorophores, utilizing radical anion states of the acceptor. In proof-of-principle experiments, we demonstrate a significant resolution increase using probes with different numbers of acceptor fluorophores. Quantitative description by a theoretical model paves the way for the development of fluorescent probes that can more than double the resolution of essentially any confocal microscope in all three dimensions.
authors.affiliation: Angewandte Physik-Biophysik, and Center for NanoScience, Ludwig-Maximilians-Universität, Amalienstrasse 54, 80799 Munich, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Vogelsang
authors.forename: Thorben
authors.initials: T
authors.lastname: Cordes
authors.forename: Carsten
authors.initials: C
authors.lastname: Forthmann
authors.forename: Christian
authors.initials: C
authors.lastname: Steinhauer
authors.forename: Philip
authors.initials: P
authors.lastname: Tinnefeld
mesh_terms: D000818:Animals; D000838:Anions; D001703:Biophysics / Q000379:methods; D001710:Biotin / Q000737:chemistry; D002417:Cattle; D004735:Energy Transfer; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008027:Light; D018613:Microscopy, Confocal / Q000379:methods*; D015233:Models, Statistical; D016011:Normal Distribution; D012710:Serum Albumin, Bovine / Q000737:chemistry
chemical_list: D000838:Anions; D005456:Fluorescent Dyes; D012710:Serum Albumin, Bovine; D001710:Biotin