title: Quantitative single-molecule imaging by confocal laser scanning microscopy.
abstract: A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.
authors.affiliation: Department of Clinical Neuroscience, Karolinska Institutet, 17176 Stockholm, Sweden. vladana.vukojevic@ki.se
authors.forename: Vladana
authors.initials: V
authors.lastname: Vukojevic
authors.forename: Marcus
authors.initials: M
authors.lastname: Heidkamp
authors.forename: Yu
authors.initials: Y
authors.lastname: Ming
authors.forename: Björn
authors.initials: B
authors.lastname: Johansson
authors.forename: Lars
authors.initials: L
authors.lastname: Terenius
authors.forename: Rudolf
authors.initials: R
authors.lastname: Rigler
mesh_terms: D004247:DNA / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes; D007834:Lasers; D018613:Microscopy, Confocal / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D004247:DNA