3D Imaging With Moving Fringe Structured Illumination Microscopy

Abstract

Structured Light Illumination Microscopy (SIM) has proved itself as a very effective method to improve the resolution of a widefield (WF) fluorescent microscope. In this paper, we demonstrate a new approach to three-dimensional (3D) imaging with the SIM, using a moving fringe (MF) illumination pattern. Instead of the standard three-beam standing wave illumination pattern, our method requires a two-beam one, varying along the optical axis. Each axial layer of the MF illumination pattern contains single-spatial-frequency interference fringes, traversing the space with its own speed (temporal frequency), proportional to the axial offset of such layer from the excitation plane. The different axial layers of a fluorescent object, excited with the MF illumination, will emit a continuous amplitude modulated fluorescent signal with the frequency of modulation proportional to the temporal frequency of the moving fringe pattern. The fine 3D image reconstruction is achieved via extracting the spatial location of the fluorescent object from the temporal frequency of amplitude modulated signal emitted by it. Since in our approach the problem of 3D image reconstruction is reduced to the problem of accurate temporal frequency estimation, any of the well-known spectrum estimation techniques can be applied to the problem, allowing the axial resolution improvement far beyond the limits of the classical 3D SIM. In this research, we suggest using the Minimum-norm method for the proposed MF SIM system, which gives a superior resolving power in spectrum estimation. Simulation results show that such a simple and rapid hardware implementation in combination with a straightforward signal processing method can, however, deliver an improvement in axial resolution far beyond the classical 3D SIM approach.