Axial Resolution Enhancement of Optical Sectioning Structured Illumination Microscopy Based on Three-Beam Interference

Abstract

As a branch of 3D microscopy, optical sectioning structured illumination microscopy (OS-SIM) has the advantages of fast imaging speed, weak photobleaching and phototoxicity, and flexible and compatible configuration. Although the method of using the one-dimensional periodic fringe pattern projected on the sample can remove the out-of-focus background from the in-focus signal, the axial resolution of the final reconstructed 3D image is not improved. Here, we propose a three-beam interference OS-SIM, namely TBOS, instead of the common-used dual-beam interference OS-SIM (DBOS). The three-beam interference scheme has been adopted in 3D super-resolution SIM (3D-SR-SIM), where the fringe phase shifting needs to be along each of the three orientations. In contrast, TBOS applies phase shifting only in one arbitrary direction. We built a TBOS SIM microscope and performed the 3D imaging experiments with 46 nm diameter fluorescent microspheres and a mouse kidney section. The axial resolution of the 3D image obtained with TBOS was enhanced by a factor of 1.36 compared to the DBOS method, consistent with the theoretical analysis and simulation. The OS-SIM with enhanced axial resolution for 3D imaging may find a wide range of applications in the biomedical field.

Keywords

• structured illumination microscopy
• optical sectioning
• three-beam interference
• axial resolution
• 3D microscopy