Enhancing Optical Sectioning in Structured Illumination Microscopy With Axially Confined Fringe Modulation

Abstract Optical sectioning structured illumination microscopy (OS‐SIM) is a fast, minimally invasive 3D imaging technique that has found widespread application in the biosciences. It is based on sample illumination with several illumination fringe patterns featuring distinct mutual phase shifts, from which an axially sectioned image is reconstructed. Its optical sectioning capability is commonly attributed to the attenuation of the fringe modulation of light collected from planes displaced from the focal plane. However, in addition to this effect, which is governed solely by the detection optics, optical sectioning can be further enhanced by confining the fringe modulation axially via partially coherent illumination (PCI). To establish guidelines for optimal illumination field shaping, both theoretically and experimentally are investigated, the optical sectioning strength of OS‐SIM upon variation of the two key parameters, modulation period and angular spectrum of the incident illumination. By using PCI with OS‐SIM, nearly fivefold and 1.4‐fold enhanced axial resolution are achieved for scattering (non‐fluorescent) and fluorescent samples, respectively. This work elucidates the optical sectioning mechanism of OS‐SIM and provides a perspective for further optimization.