Single-molecule orientation mapping in 3D using a novel optical design

Single molecule (SM) localization-based super-resolution techniques have revolutionized the application of optical microscopy because of their ability to image structures below the diffraction limit in widefield. Fluorescent dyes and proteins used for SM imaging absorb and emit photons via an emission dipole, and any restriction on the motion of the dipole creates an asymmetry in the observation of the emission. Mapping this dipolar orientation can be a useful reporter of the local environment of molecules, and help improve localization accuracy for super-resolution imaging. We present a simple and flexible optical design, that can be incorporated into a standard optical microscope, to enable mapping the orientation of single molecules by simultaneously imaging 4 polarization orientations. A single camera is used to acquire all 4 channels, and fine adjustment controls for each channel minimize optical aberrations. We have used nanofabricated aperture arrays, fluorescent beads and single molecules embedded in a polymer matrix to characterize the system.