Light sheet microscopy is a powerful technique for visualizing dynamic biological processes in 3D. Studying large specimens or recording time series with high spatial and temporal resolution generates large datasets, often exceeding terabytes and potentially reaching petabytes in size. Handling these massive datasets is challenging for conventional data processing tools with their memory and performance limitations. To overcome these issues, we developed LLSM5DTools, a software solution specifically designed for the efficient management of petabyte-scale light sheet microscopy data. This toolkit, optimized for memory and per-formance, features fast image readers and writers, efficient geometric transformations, high-performance Richardson-Lucy deconvolution, and scalable Zarr-based stitching. These advancements enable LLSM5DTools to perform over ten times faster than current state-of-the-art methods, facilitating real-time processing of large datasets and opening new avenues for biological discoveries in large-scale imaging experiments.

Chemigenetic tags are versatile labels for fluorescence microscopy that combine some of the advantages of genetically encoded tags with small molecule fluorophores. The Fluorescence Activating and absorbance Shifting Tags (FASTs) bind a series of highly fluorogenic and cell-permeable chromophores. Furthermore, FASTs can be used in complementation-based systems for detecting or inducing protein-protein interactions, depending on the exact FAST protein variant chosen. In this study, we systematically explore substitution patterns on FAST fluorogens and generate a series of fluorogens that bind to FAST variants, thereby activating their fluorescence. This effort led to the discovery of a novel fluorogen with superior properties, as well as a fluorogen that transforms splitFAST systems into a fluorogenic dimerizer, eliminating the need for additional protein engineering.