Silvestri, L. et al. (2021) Universal autofocus for quantitative volumetric microscopy of whole mouse brains. Nature Methods, 18(8), pp. 953-958. (doi: 10.1038/s41592-021-01208-1) (PMID:34312564)
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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.
| Item Type: | Articles |
|---|---|
| Additional Information: | 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 Skłodowska-Curie fellowship (MSCA-IF-EF-ST grant agreement no. 842893). |
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Muellenbroich, Dr Caroline |
| Authors: | Silvestri, L., Müllenbroich, M. C., Costantini, I., Di Giovanna, A. P., Mazzamuto, G., Franceschini, A., Kutra, D., Kreshuk, A., Checcucci, C., Toresano, L. O., Frasconi, P., Sacconi, L., and Pavone, F. S. |
| College/School: | College of Science and Engineering > School of Physics and Astronomy |
| Journal Name: | Nature Methods |
| Publisher: | Nature Research |
| ISSN: | 1548-7091 |
| ISSN (Online): | 1548-7105 |
| Published Online: | 26 July 2021 |
| Copyright Holders: | Copyright © 2021 The Authors |
| First Published: | First published in Nature Methods 18(8): 953-958 |
| Publisher Policy: | Reproduced in accordance with the publisher copyright policy |
| Related URLs: | |
| Data DOI: | 10.25493/AV5J-M46 |
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