title: Identification of immobile single molecules using polarization-modulated asynchronous time delay and integration-mode scanning.
abstract: We report the development of a data acquisition method for identifying single molecules on large surfaces with simultaneous characterization of their absorption dipole. The method is based on a previously described device for microarray readout at single molecule sensitivity (Hesse, J.; Sonnleitner, M.; Sonnleitner, A.; Freudenthaler, G.; Jacak, J.; Höglinger, O.; Schindler, H.; Schütz, G. J. Anal. Chem. 2004, 76, 5960-5964). Here, we introduced asynchronous time delay and integration- (TDI-) mode imaging to record also the time course of fluorescence signals: the images thus contain both spatial and temporal information. We demonstrate the principle by modulating the signals via rotating excitation polarization, which allows for discriminating static absorption dipoles against multiple or freely rotating single absorption dipoles. Experiments on BSA carrying different numbers of fluorophores demonstrate the feasibility of the method. Protein species with an average labeling degree of 0.55 and 2.89 fluorophores per protein can be readily distinguished.
authors.affiliation: Biophysics Institute, Johannes Kepler University Linz, Altenberger Strasse 69, 4040 Linz, Austria.
authors.forename: Jaroslaw
authors.initials: J
authors.lastname: Jacak
authors.forename: Clemens
authors.initials: C
authors.lastname: Hesch
authors.forename: Jan
authors.initials: J
authors.lastname: Hesse
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Schütz
mesh_terms: D004867:Equipment Design; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D036103:Nanotechnology / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000379:methods*; D018511:Systems Integration; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D012710:Serum Albumin, Bovine