Systematic and Quantitative Comparison of Axially Overlapped Multi-Focus and Gaussian Light Sheets

Overlapped multi-focus light sheet (MF-LS) along beam propagation microscope has generated significant interest due to its reportedly superior axial resolving power when compared to conventional Gaussian light sheet (GLS) systems. By axially overlapped multiple light sheets in the illumination path, it would weld the combined field of view (FOV) and be wider than stationary counterparts. However, the effect of diffractive energy entering the focal space of desired beam, impacting the optical sectioning capabilities of the system has not been studied. It introduces the question that does a MF-LS performance better than standard GLS? Here we quantitatively and systematically analyzed the properties between optimized MF-LS and traditional GLS on z-axis resolution, signal to background ratio (SBR), and FOV. We first optimized the MF-LS by analyzing the diffractive energy to acquire the best sectioning capability. Then, we demonstrated a comparable optical sectioning performance between the two types of LS when matching with specific optical parameters including the illumination numerical aperture (NA) and foci numbers. A novel equation has been proposed to describe the equivalence relation. Analysis in the research suggests “putative FOV-extension” characteristics of MF-LS due to energy effects and interference between beams. Finally, we performed simulation and imaging experiments approaches on both fluorescence microspheres and biological samples to validate the viewpoint of this research.