Characterization, comparison, and optimization of lattice light sheets

Lattice light sheet microscopy excels at the noninvasive imaging of three-dimensional (3D) dynamic processes at high spatiotemporal resolution within cells and developing embryos. Recently, several papers have called into question the performance of lattice light sheets relative to the Gaussian sheets most common in light sheet microscopy. Here, we undertake a theoretical and experimental analysis of various forms of light sheet micros-copy, which demonstrates and explains why lattice light sheets provide substantial improvements in resolution and photobleaching reduction. The analysis provides a procedure to select the correct light sheet for a desired experiment and specifies the processing that maximizes the use of all fluorescence generated within the light sheet excitation envelope for optimal resolution while minimizing image artifacts and photodamage. We also introduce a new type of harmonic balanced lattice light sheet that improves performance at all spatial fre-quencies within its 3D resolution limits and maintains this performance over lengthened propagation distances allowing for expanded fields of view.

Automated summary

Lattice light sheet microscopy is excellent for noninvasive imaging of 3D dynamic processes within cells and embryos. However, its performance has recently been questioned compared to Gaussian sheets commonly used in light sheet microscopy. This study presents a theoretical and experimental analysis of different types of light sheet microscopy, showing that lattice light sheets offer significant improvements in resolution and photobleaching reduction. It also provides guidelines for selecting the appropriate light sheet and optimizing fluorescence usage for optimal resolution and minimal image artifacts and photodamage. A new type of harmonic balanced lattice light sheet is introduced, which improves performance across all spatial frequencies and allows for expanded fields of view.