Two-step phase-shifting sectioning structured illumination microscopy

Two-step phase-shifting sectioning structured light illumination microscopy (TSSIM) that reconstructs a three-dimensional structure using Fourier transform is proposed. Undesirable background signals corresponding to out-of-focus signals are eliminated using this method. Since there is no restriction for accurate phase shifts, this method does not suffer from large retrieval errors, unlike conventional sectioning structured illumination wide-field fluorescence microscopy (SSIWM). It can be used directly without modifying the conventional SSIWM microscope setup employing two of the three captured images, and can be applied to both shape measurements and biological observation. Less exposure time is required; thus, photobleaching and phototoxicity in biological observation are reduced. Further, the impact of the phase-shift difference on the signal-to-noise ratio of reconstruction image is analyzed. Both simulations and experiments are presented to show the validity of the proposed method.