A quantitative analysis of various patterns applied in lattice light sheet microscopy

Abstract

AbstractLight sheet microscopes reduce phototoxicity and background and improve imaging speed compared to widefield and confocal microscopes. However, when equipped with Gaussian beams, the axial resolving power of a light sheet microscope and the observable field of view are inversely related. Light sheets based on dithered optical lattices improve axial resolution and beam uniformity compared Gaussian beams by using axially structured illumination patterns. However, these advantages come at the expense of an increased total illumination to the specimen and a decreased axial confinement of the illumination pattern. Using simulations and experimental measurements in fixed and live cells, we quantify the differences between Gaussian and lattice light sheets on beam uniformity, axial resolution, lateral resolution, and photobleaching. We demonstrate how different optical lattice illumination patterns can be tuned to prioritize either axial resolution or optical sectioning. Finally, we introduce an approach to spectrally fuse sequential acquisitions of different lattice light sheet patterns with complementary optical properties to achieve both high resolution and low background images.