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.