Overview of Common Spectroscopic Methods to Determine the Orientation/Alignment of Membrane Probes and Drugs in Lipidic Bilayers

Abstract

The in-depth location and orientation of membrane probes and drugs inserted in lipidic bilayers are regarded important key-properties that cannot be overlooked during molecular design and synthesis. Several spectroscopic phenomena (e.g. excitonic interaction) and molecular recognition (e.g. ligand-receptor interaction) depend on these properties. However, molecular orientation in lipidic membranes is scarcely addressed. This paper overviews some of the most important techniques and methodologies used to study orientation of molecules relative to the surrounding lipidic matrix, namely: FTIR linear dichroism, UV-Vis linear dichroism, Time-resolved fluorescence anisotropy, NMR, and Surface Plasmon Resonance.

Combinatorial microscopy for the study of protein–membrane interactions in supported lipid bilayers: Order parameter measurements by combined polarized TIRFM/AFM

“…This quantity is the fluorescence-detected analog of the absorption dichroic ratio seen in polarized attenuated total internal reflection Fourier transform infrared spectroscopy (pATIR-FTIR) Citra and Axelsen, 1996;Lopes and Castanho, 2005;Tamm and Tatulian, 1997). An R FD image is formed simply by pixelwise division of the acquired F p and F s images.…”

“…The technique of polarized total internal reflection fluorescence microscopy (pTIRFM) is able to infer the time-and ensemble-averaged orientational order related to a collection of fluorescent probe molecules located within an interfacial system such as a substrate-supported bilayer through a quantity known as the orientational order parameter, hP 2 i (Axelrod, 1989(Axelrod, , 2001Burghardt, 1984;Oreopoulos and Yip, 2009;Sund et al, 1999;Thompson et al, 1984;Timbs and Thompson, 1990). This order parameter is similar to those encountered in other orientation-sensitive spectroscopies and has an analogous interpretation Bolterauer and Heller, 1996;Douliez et al, 1998;Lafrance et al, 1995;Lopes and Castanho, 2005;Schafer et al, 1998;Vermeer et al, 2007). In addition to the co-localization of sample fluorescence and topography that can be achieved by combined optical and atomic force microscopy, pTIRFM also allows hP 2 i associated with the fluorescent probes to be spatially mapped via the (probe concentration-independent) order parameter image.…”

Simple Estimation of Förster Resonance Energy Transfer (FRET) Orientation Factor Distribution in Membranes

“…Even though computational calculations of κ 2 are scarce, determination of membrane-embedded fluorophore orientations using computational (for reviews of MD studies of fluorescent membrane probes, see [11,12]) or experimental techniques (traditionally linear dichroism or other spectroscopic methods [13,14], or, more recently, methods based on polarized total internal reflection fluorescence microscopy or other microscopic techniques [15–17]) are more frequent.…”

Fluorescence Anisotropy to Study the Preferential Orientation of Fluorophores in Ordered Bi-Dimensional Systems: Rhodamine 6G/Laponite Layered Films

“…1). Similar to the absorption spectroscopy with linearly polarized light, the fluorescence intensity will depend on the relative orientation of the emission transition moment of the fluorophore ( M) and the direction of the emission polarizer [80,82,[86][87][88][89][90], concretely proportional to the square of the projection of M in the direction of the polarizer, Eqs. 1 and 2.…”