We introduce single molecule light field microscopy (SMLFM), a novel 3D single molecule localization technique that is capable of up to 20 nm isotropic precision across a 6 μ m depth of field. SMLFM can be readily implemented by installing a refractive microlens array into the conjugate back focal plane of any widefield single molecule localization system. We demonstrate that 3D localization can be performed by post-processing 2D localization data generated by common, widely-used, algorithms. In this work we benchmark the performance of SMLFM and finally showcase its capabilities by imaging fluorescently labeled membranes of fixed eukaryotic cells below the diffraction limit.

Single Molecule Light Field Microscopy