3D Optical Sectioning Microscopy with Sparse Structured Illumination

Optical microscopy has the advantages of low sample damage as well as high specificity imaging and is an indispensable imaging technique in many fields, such as biomedicine, life science, and material chemistry. However, the conventional optical microscope uses parallel light to illuminate the entire sample, which cannot effectively distinguish the in-focus signal from the defocused background, and does not have the ability of realizing a three-dimensional optical section imaging. Hence, a sparse structured illumination three-dimensional sectioning microscopy (SSI-3DSM) technology based on resonant scanning is proposed herein. The sparse structured illumination is rapidly generated via the resonant scanning focusing spot, and the three-dimensional optical section imaging of the sample to be measured is realized using a multistep phase shift to determine and eliminate the background noise. Compared with scanning wide-field imaging, the proposed method improves the axial resolution by 1. 3 times and the signal-to-background ratio by 12 times. Moreover, the proposed method demonstrates stable performance, is cost effective, and can be easily commercialized. It can be combined with super-resolution microscopy imaging techniques such as structured illumination microscopy and single-molecule localization to further improve lateral resolution.