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bioRxiv · 10.1101/2022.07.30.502108

Characterization, Comparison, and Optimization of Lattice Light Sheets

Ruan, X.·Liu, G.·Milkie, D. E.·Gorlitz, F.·Mueller, M.·Hercule, W.·Kililea, A.·Betzig, E.·Upadhyayula, S.
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Abstract

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. Significance StatementDespite its rapidly growing use, several misconceptions remain concerning the physics of image formation and its optimization in light sheet microscopy, particularly in high resolution variants tailored for subcellular imaging. These include the role of excitation sidelobes, the significance of out-of-focus fluorescence, the importance and optimization of deconvolution, and the perceived advantages of Gaussian beams. Here we attempt to shatter these misconceptions by showing that the professed tradeoffs between axial resolution and background haze, photobleaching rate, phototoxicity, and propensity for image artifacts do not exist for well-crafted lattice light sheets whose data is acquired and processed rigorously. The framework we provide should enable others to optimize light sheets and extract the most information at the lowest cost in their experiments.

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Ruan, X., Liu, G., Milkie, D. E., Gorlitz, F., Mueller, M., Hercule, W., Kililea, A., Betzig, E., Upadhyayula, S.. 2022-07-30. Characterization, Comparison, and Optimization of Lattice Light Sheets. https://doi.org/10.1101/2022.07.30.502108

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