Vortex light field microscopy: 3D spectral single-molecule imaging with a twist

3D single-molecule imaging reveals nanoscale structures in cell volumes but is limited by the need for spectrally distinct fluorophores. We address this limitation with vortex light field microscopy (VLFM), a 3D spectral single-molecule localization technique with 25 nm spatial and 3 nm spectral precision over a 4 µm depth of field. By modifying our previous single-molecule light field microscope with an azimuthally oriented prism array, we generated spectral disparity orthogonal to axial disparity, enabling simultaneous spatial and spectral localization on a single detector. We demonstrate VLFM with four-color 3D single-particle tracking and two-color 3D dSTORM imaging in fixed cells, successfully identifying dyes with spectral peaks just 15 nm apart. This shows VLFM’s potential for enhancing spatial biology workflows requiring highly multiplexed imaging.