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.

Simulations of camera-based single-molecule fluorescence experiments

“…The probability of n ph incident photons to be detected on the detector pixel ( x , y ) during a frame l is given by a Poisson distribution P ( n ph ; μ ph = I ( x , y , l ) D/A characterized by the mean number of incident photons μ ph . The simulation of sole photon noise is commonly used for the simulation of single photon detection applications [ 19 , 45 , 59 – 62 ]. (ii) The second noise contribution is the camera shot noise σ pe common to all (EM)CCDs or sCMOS [ 42 , 43 ].…”

An atomistic view on carbocyanine photophysics in the realm of RNA

“…All these parameters are readily accessible by time-resolved or steady-state measurements and report on the physicochemical characteristics of the fluorophore within its anisotropic, macromolecular environment. 24 The photophysics of carbocyanines in the context of DNA are well established, 12,[25][26][27][28] however, our knowledge about the interaction propensity with RNA on an atomic level remains elusive. Recent work has demonstrated the applicability of protein induced fluorescence enhancement (PIFE) as a spectroscopic ruler to close the gap between the distance regimes covered by photoinduced electron transfer (PET, o2 nm) and FRET (3-10 nm).…”

Enhanced DNA Imaging Using Super-Resolution Microscopy and Simultaneous Single-Molecule Orientation Measurements

“…In order to measure either of these two parameters without incurring systematic bias, it is essential to measure polarized single-molecule intensity along a minimum of three unique polarization axes. (See e. g. [28][29][30][31] for alternative schemes.) Furthermore, dye molecules may rapidly rotate or wobble [32][33][34], emitting fluorescence at many distinct orientations.…”