title: Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes.
abstract: Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels. As the method is based on the detection of single photons, it additionally allows for performing fluorescence correlation spectroscopy (FCS) as well as dynamical anisotropy measurements thereby providing access to fast orientational dynamics down to the nanosecond time scale. The 3D orientation is particularly interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination at different timescales and quantifying the associated errors. The vesicles provide a well-defined spherical surface, such that the use of fluorescent lipid dyes (DiO) allows to establish a a wide range of dipole orientations experimentally. To complement our experimental data, we performed Monte Carlo simulations of the rotational dynamics of dipoles incorporated into lipid membranes. Our study offers a comprehensive view on the dye orientation behavior in a lipid membrane with high spatiotemporal resolution representing a six-dimensional fluorescence detection approach.
authors.affiliation: Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland. richard.boerner@uzh.ch.
authors.forename: Richard
authors.initials: R
authors.lastname: Börner
authors.affiliation: Bionanotechnology and Nanomedicine Laboratory, University of Copenhagen, Universitetsparken 5, 2100, Copenhagen, Denmark.
authors.forename: Nicky
authors.initials: N
authors.lastname: Ehrlich
authors.affiliation: Laboratory of Biophysics, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands.|Microspectroscopy Centre, Wageningen University, 6703 HA, Wageningen, The Netherlands.
authors.forename: Johannes
authors.initials: J
authors.lastname: Hohlbein
authors.affiliation: Institute of Physics, University of Lübeck, Ratzeburger Allee 160, 23562, Lübeck, Germany.
authors.forename: Christian G
authors.initials: CG
authors.lastname: Hübner
keywords: 3D dipole orientation; Anisotropy; Confocal microscopy; FCS; Fluorescence microscopy; GUV; Single molecule