This PAINT dataset is a movie of the fluorescence of single Nile red molecules reversibly binding to a lipid bilayer (DPPC + 40% cholesterol) on a 5 μm diameter silica microsphere (44054-5ML-F, Sigma-Aldrich). Images were recorded at 3 different focal planes; approximately at the midpoint of the bead (z1), the top of the bead (z3) and a position in between (z2). The data was collected on a fluorescence microscope (Eclipse Ti2, Nikon) with a polarisation camera (CS505MUP, Thorlabs). The molecules were excited with a 514 nm DPSS laser with a measured power density at the sample plane of 6.08 kW/cm^2. An exposure time of 50 ms was used. The following filters were used: dichroic (Di03-R514-t1, Semrock) and emission filter (FF01-515/LP and FF01-650/200, Semrock).