Universal autofocus for quantitative volumetric microscopy of whole mouse brains

Year: 2021

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 (8)      Pages from: 953  to: +

More Information: The authors thank F. Del Gallo from the University of Padova (Italy) for providing mouse brain with labeled microglia, I. Tolic from the Ruder Boskovic Institute (Croatia) for providing the fluorescent Schizosaccharomyces pombe, and R. Cicchi from the National Institute of Optics (Italy) for providing the histological slides used in this study. This project received funding from the European Union’s Horizon 2020 research and innovation program under grant agreements no. 785907 (Human Brain Project) and 871124 (Laserlab-Europe), and from the H2020 EU program Excellent Science in conjunction with the European Research Council (ERC) under grant agreement ID no. 692943 (BrainBIT). The project has also been supported by the Italian Ministry for Education, University, and Research within the framework of the Flagship Project NanoMAX and of the Advance Lightsheet Microscopy Italian Mode of Euro-Bioimaging ERIC, and by Ente Cassa di Risparmio di Firenze (a private foundation). M.C.M. acknowledges a Marie Sklodowska-Curie fellowship (MSCA-IF-EF-ST grant agreement no. 842893).
KeyWords: Light-sheet Microscopy; Segmentation; Neurons; Cells
DOI: 10.1038/s41592-021-01208-1

ImpactFactor: 47.990
Citations: 31
data from “WEB OF SCIENCE” (of Thomson Reuters) are update at: 2024-11-24
References taken from IsiWeb of Knowledge: (subscribers only)

Connecting to view paper tab on IsiWeb: Click here
Connecting to view citations from IsiWeb: Click here