title: High-resolution colocalization of single molecules within the resolution gap of far-field microscopy.
abstract: To obtain detailed information about the three-dimensional (3D) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 630/650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 630/650) that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.
authors.affiliation: Institute of Physical Chemistry, University of Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Heinlein
authors.forename: Andreas
authors.initials: A
authors.lastname: Biebricher
authors.forename: Pia
authors.initials: P
authors.lastname: Schlüter
authors.forename: Christian Michael
authors.initials: CM
authors.lastname: Roth
authors.forename: Dirk-Peter
authors.initials: DP
authors.lastname: Herten
authors.forename: Jürgen
authors.initials: J
authors.lastname: Wolfrum
authors.forename: Mike
authors.initials: M
authors.lastname: Heilemann
authors.forename: Christian
authors.initials: C
authors.lastname: Müller
authors.forename: Philip
authors.initials: P
authors.lastname: Tinnefeld
authors.forename: Markus
authors.initials: M
authors.lastname: Sauer
mesh_terms: D000465:Algorithms; D001703:Biophysics / Q000379:methods*; D001896:Boron Compounds / Q000494:pharmacology; D002232:Carbocyanines / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D004583:Electrons; D020549:GTP Phosphohydrolase Activators / Q000737:chemistry; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008853:Microscopy / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D016011:Normal Distribution; D012984:Software; D013997:Time Factors
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D020549:GTP Phosphohydrolase Activators; D004247:DNA