Total internal reflection fluorescence based multiplane localization microscopy enables super-resolved volume imaging

摘要

Total internal reflection fluorescence (TIRF) based geometry is attractive for super-resolution localization microscopy. Although the traditional TIRF configuration enables near-surface 2D imaging, it is not capable of imaging multiple axial planes. We propose a simultaneous multiplane imaging based localization encoded (SMILE) technique in the TIRF configuration that utilizes point spread function (PSF) information (PSF size, corresponding to single molecules located at the focal plane and off-focal planes, and the detected photons per PSF) to reconstruct a near-surface volume stack. The natural spread of the detection PSFs (far from the specimen-coverslip interface) is used to fix the axial locations of single molecules, and the corresponding photon count determines their localization precision. The proposed SMILE microscopy technique enables super-resolved volume reconstruction based on 2D recorded data.