Mapping the Chemical Composition and Nanoscale Structure of Lipid Membranes Using Polarized Fluorescence from Single Molecules

Single-molecule orientation localization microscopy (SMOLM) provides insight into biological activities that cannot be resolved using standard superresolution techniques. Here, we present a radially and azimuthally polarized (raPol) microscope for tracking the 2D positions and 3D orientations of fluorescent molecules. This technique is simply implemented by adding a commercially available vortex half waveplate and polarizing beamsplitter to an epifluorescence microscope. Compared to other SMOLM techniques, the raPol microscope exhibits superior detectability and excellent measurement precision, which enables us to resolve the chemical composition and nanoscale structure of lipid membranes.