Journal of Fluorescence

, Volume 26, Issue 3, pp 963–975 | Cite as

Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes

  • Richard BörnerEmail author
  • Nicky Ehrlich
  • Johannes Hohlbein
  • Christian G. Hübner


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.


Single molecule Fluorescence microscopy 3D dipole orientation Confocal microscopy GUV Anisotropy FCS 


Funding and Acknowledgement

The Volkswagen Stiftung (AZ I/83 019 to CGH), a Marie Curie Career Integration Grant (#630992 to JH) and the Forschungskredit grant of the University Zurich (FK-14-096, to RB) are gratefully acknowledged for financial support. We thank Henning Seidel for the implementation of the 3D sectioning scanning in the Labview based setup control software of our home-built SCOM and Sebastian L.B. König, University of Zurich, for proofreading the manuscript.

Author Contributions

Conceived and designed the experiments: RB NE JH CGH. Performed the experiments: RB. Analyzed the data: RB. Contributed reagents/materials: RB NE. Wrote the paper: RB JH NE CGH. Derived mathematical descriptions: RB. Designed and programmed software: RB JH.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

10895_2016_1784_MOESM1_ESM.doc (1.5 mb)
ESM 1 (DOC 1539 kb)


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Richard Börner
    • 1
    Email author
  • Nicky Ehrlich
    • 2
  • Johannes Hohlbein
    • 3
    • 5
  • Christian G. Hübner
    • 4
  1. 1.Department of ChemistryUniversity of ZurichZurichSwitzerland
  2. 2.Bionanotechnology and Nanomedicine LaboratoryUniversity of CopenhagenCopenhagenDenmark
  3. 3.Laboratory of BiophysicsWageningen UniversityWageningenThe Netherlands
  4. 4.Institute of PhysicsUniversity of LübeckLübeckGermany
  5. 5.Microspectroscopy CentreWageningen UniversityWageningenThe Netherlands

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