3D structured illumination microscopy using an incoherent illumination system based on a Fresnel biprism

Abstract

Three-dimensional (3D) structured illumination (SI) patterns that include lateral and axial variations have attracted more attention recently as their use in fluorescence microscope enhances the 3D resolution of the native imaging system. 3D SI patterns have already been created by interfering three mutually-coherent waves using a diffraction grating or some electro-optical devices such as spatial light modulators. Here, an interesting approach to generate a 3D SI pattern of tunable modulation frequency is shown. Our proposed illumination system is based on the incoherent illumination of a Fresnel biprism using several equidistant linear sources (i.e., slits). Previously, we investigated and compared numerically this tunable SI microscopy (SIM) system with the one achieved with three-wave interference. In this contribution, we implement our proposed incoherent 3D SIM system of tunable-frequency in an open-setup. We evaluate the axial confinement of the illumination pattern obtained with this system by recording the SI pattern using a mirror sample and different number of slits and compare these data with simulation results. Moreover, we verify that with a higher number of slits used, the axial confinement of the pattern increases, and consequently, the system’s optical sectioning capability improves.