Simultaneous multiplane imaging‐based localization encoded (SMILE) microscopy for super‐resolution volume imaging

We propose a widefield‐based rapid super‐resolution volume imaging technique. This technique requires encoding single molecules to their respective planes and subsequent identification of the locus of individual molecule (both in the focal plane and off‐focal planes). Experimentally, this is achieved by precise calibration of system PSF size and its natural spread in the off‐focal planes using sub‐diffraction fluorescent beads. The specimen plane touching the coverslip is chosen as the focal plane whereas planes far from coverslip (situated at large penetration depths) represent off‐focal planes. The identification and sorting of single molecules are carried out by setting multiple cut‐offs to the respective PSFs and a 3D super‐resolved volume image is reconstructed. SMILE microscopy technique eliminates the need for multiple z‐plane scanning, minimizes radiation‐dose and enables rapid super‐resolution volume imaging.