Lattice light sheet microscopy excels at the non-invasive 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 comprehensive theoretical and experimental analysis of various forms of light sheet microscopy which both demonstrates and explains why lattice light sheets provide significant 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. Development of a new type of “harmonic balanced” lattice light sheet is shown to improve performance at all spatial frequencies within its 3D resolution limits and maintains this performance over lengthened propagation distances allowing for expanded fields of view.

The dataset was collected with a homemade lattice light sheet microscope with different light sheets and different parameters. The light sheet name and parameters are contained in the zip file names. 

The dataset has been processed with LLSM5DTools (https://github.com/abcucberkeley/LLSM5DTools/tree/dev). 

The tiff files can be opened with any 3D visualization software (i.e., Fiji, napari, Imaris and Amira) with the support of the 3D tiff format.