title: The Effect of a Fluorophore Photo-Physics on the Lipid Vesicle Diffusion Coefficient Studied by Fluorescence Correlation Spectroscopy.
abstract: Fluorescence Correlation Spectroscopy (FCS) is a technique, which allows determination of the diffusion coefficient and concentration of fluorescent objects suspended in the solution. The measured parameter is the fluctuation of the fluorescence signal emitted by diffusing molecules. When 100 nm DOPC vesicles labeled with various fluorescent dyes (Fluorescein-PE, NBD-PE, Atto488 DOPE or βBodipy FL) were measured, different values of diffusion coefficients have been obtained. These diffusion coefficients were different from the expected values measured using the dynamic light scattering method (DLS). The FCS was initially developed for solutions containing small fluorescent molecules therefore the observed inconsistency may result from the nature of vesicle suspension itself. The duration of the fluorescence signal may depend on the following factors: the exposure time of the labeled object to the excitation beam, the photo-physical properties (e.g., stability) of a fluorophore, the theoretical model used for the calculations of the diffusion coefficient and optical properties of the vesicle suspension. The diffusion coefficients determined for differently labeled liposomes show that its dependence on vesicle size and quantity of fluorescent probed used for labeling was significant demonstrating that the fluorescence properties of the fluorophore itself (bleaching and/or blinking) were critical factors for a correct outcome of FCS experiment. The new, based on combined FCS and DLS measurements, method for the determination of the focal volume prove itself to be useful for the evaluation of a fluorescence dye with respect to its applicability for FCS experiment.
authors.affiliation: Laboratory for Biophysics of Macromolecular Aggregates, Department of Biomedical Engineering, Wroclaw University of Technology, Pl. Grunwaldzki 13, 50-377, Wroclaw, Poland. Dominik.Drabik@pwr.edu.pl.
authors.forename: Dominik
authors.initials: D
authors.lastname: Drabik
authors.affiliation: Laboratory for Biophysics of Macromolecular Aggregates, Department of Biomedical Engineering, Wroclaw University of Technology, Pl. Grunwaldzki 13, 50-377, Wroclaw, Poland.|Lipid Systems sp. z. o. o. [Ltd], ul. Duńska 9, 54-066, Wrocław, Poland.
authors.forename: Magda
authors.initials: M
authors.lastname: Przybyło
authors.affiliation: Laboratory of Cytobiochemistry, Faculty of Biotechnology, University of Wroclaw, ul. Joliot-Curie 14a, Wrocław, Poland.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Sikorski
authors.affiliation: Laboratory for Biophysics of Macromolecular Aggregates, Department of Biomedical Engineering, Wroclaw University of Technology, Pl. Grunwaldzki 13, 50-377, Wroclaw, Poland.|Lipid Systems sp. z. o. o. [Ltd], ul. Duńska 9, 54-066, Wrocław, Poland.
authors.forename: Marek
authors.initials: M
authors.lastname: Langner
mesh_terms: D004058:Diffusion; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D005997:Glycerylphosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D008962:Models, Theoretical; D010713:Phosphatidylcholines; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D008081:Liposomes; D010713:Phosphatidylcholines; D005997:Glycerylphosphorylcholine; C017251:1,2-oleoylphosphatidylcholine
keywords: Blinking; Dynamic light scattering; Fluorescence correlation spectroscopy; Fluorescent probes; Photo-bleaching