Evaluating structured-illumination patterns in optimizing optical-sectioning of HiLo microscopy

Abstract

HiLo microscopy is a wide-field, optical-sectioning imaging technique, which is based on computational reconstruction from a uniform-illumination image and a structured-illumination (SI) image. Considering different imaging situations, the parameters of SI patterns should be optimized. However, no such discussion has been presented in grid-pattern (such as sinusoidal pattern and checkerboard pattern) based HiLo microscopy. Here, we analyze the dependence of optical-sectioning performance on parameters of SI pattern via both numerical simulations and imaging experiments. We find that both lower frequency and lower duty cycle of the grid-based pattern would introduce higher modulation depth, which would induce less artifacts in final reconstruction. Besides, at given illumination wavelength and objective numerical aperture, the higher the frequency of the grid-based pattern is, the better the axial resolution is, and thus the better the contrast of the optical-sectioning image is. Specially, the axial resolution is optimal when the frequency of pattern is set to half of cut-off frequency. We expect it as a guide of optimizing parameters of SI patterns for optical-sectioning in HiLo microscopy.