In recent advancements in life sciences, optical microscopy has played a crucial role in acquiring high-quality three-dimensional structural and functional information. However, the quality of 3D images is often compromised due to the intense scattering effect in biological tissues, compounded by several issues such as limited spatiotemporal resolution, low signal-to-noise ratio, inadequate depth of penetration, and high phototoxicity. Although various optical sectioning techniques have been developed to address these challenges, each method adheres to distinct imaging principles for specific applications. As a result, the effective selection of suitable optical sectioning techniques across diverse imaging scenarios has become crucial yet challenging. This paper comprehensively overviews existing optical sectioning techniques and selection guidance under different imaging scenarios. Specifically, we categorize the microscope design based on the spatial relationship between the illumination and detection axis, i.e., on-axis and off-axis. This classification provides a unique perspective to compare the implementation and performances of various optical sectioning approaches. Lastly, we integrate selected optical sectioning methods on a custom-built off-axis imaging system and present a unique perspective for the future development of optical sectioning techniques.
© 2025. The Author(s).