We introduce single molecule light field microscopy (SMLFM), a new class of three-dimensional (3D) single molecule localization microscopy. By segmenting the back focal plane of a microscope objective with an array of microlenses to generate multiple 2D perspective views, the same single fluorophore can be imaged from different angles. These views, in combination with a bespoke fitting algorithm, enable the 3D positions of single fluorophores to be determined from parallax. SMLFM achieves up to 20 nm localization precision throughout an extended
6
µ
m
depth of field. The capabilities of SMLFM are showcased by imaging membranes of fixed eukaryotic cells and DNA nanostructures below the optical diffraction limit.</jats:p
