Spectrally Resolved Single Molecule Orientation Imaging Reveals Direct Correspondence between Polarity and Order Experienced by Nile Red in Supported Lipid Bilayer Membrane

ABSTRACT Molecular level interaction among lipids, cholesterol and water dictates nanoscale membrane organization of lipid bilayers into liquid ordered (Lo) and liquid disordered (Ld) phases, characterized by different polarity and order. Generally, solvatochromic dyes easily discriminate polarity difference between Lo and Ld phases, whereas molecular flippers and rotors show distinct photophysics depending on membrane order. In spite of progress in single molecule spectral imaging and single molecule orientation mapping, still direct experimental proof linking polarity with order sensed by the same probe eludes us. Here, we demonstrate spectrally resolved single molecule orientation localization microscopy to connect nanoscopic localization of probe on bilayer membrane with its emission spectra, three-dimensional dipole orientation and rotational constraint offered by the local microenvironment and highlights the beautiful correspondence between polarity and order. This technique has the potential to addres nanoscale heterogeneity and dynamics, especially in biology as well as material sciences. TOC GRAPHICS