Universal autofocus for quantitative volumetric microscopy of whole mouse brains
Authors: Silvestri L., Mullenbroich MC., Costantini I., Di Giovanna AP., Mazzamuto G., Franceschini A., Kutra D., Kreshuk A., Checcucci C., Toresano LO., Frasconi P., Sacconi L., Pavone FS.
Autors Affiliation: University of Florence; European Laboratory for Nonlinear Spectroscopy (LENS); National Research Council – National Institute of Optics (CNR-INO); University of Glasgow; European Molecular Biology Laboratory (EMBL)
Abstract: Unbiased quantitative analysis of macroscopic biological samples demands fast imaging systems capable of maintaining high resolution across large volumes. Here we introduce RAPID (rapid autofocusing via pupil-split image phase detection), a real-time autofocus method applicable in every widefield-based microscope. RAPID-enabled light-sheet microscopy reliably reconstructs intact, cleared mouse brains with subcellular resolution, and allowed us to characterize the three-dimensional (3D) spatial clustering of somatostatin-positive neurons in the whole encephalon, including densely labeled areas. Furthermore, it enabled 3D morphological analysis of microglia across the entire brain. Beyond light-sheet microscopy, we demonstrate that RAPID maintains high image quality in various settings, from in vivo fluorescence imaging to 3D tracking of fast-moving organisms. RAPID thus provides a flexible autofocus solution that is suitable for traditional automated microscopy tasks as well as for quantitative analysis of large biological specimens.
Journal/Review: NATURE METHODS
Volume: 18 Pages from: 953 to: 958
KeyWords: light sheet microscopy, mouse brain, neuronal imaging, cell countingDOI: 10.1038/s41592-021-01208-1Citations: 13data from “WEB OF SCIENCE” (of Thomson Reuters) are update at: 2023-03-26References taken from IsiWeb of Knowledge: (subscribers only)Connecting to view paper tab on IsiWeb: Click hereConnecting to view citations from IsiWeb: Click here