This image dataset is a movie of the fluorescence of single SYTOX Orange molecules (S34861, Invitrogen) dispersed on a cover glass. The data was collected on a fluorescence microscope (Ti-U, Nikon) with a polarisation camera (CS505MUP, Thorlabs). The molecules were excited with a 532 nm diode laser with a measured power density at the sample plane of 0.36 kW/cm^2. An exposure time of 100 ms was used. The following filters were used: dichroic (Di03-R532-t1, Semrock) and emission filter (FF01-582/64, Semrock).