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.
Universal autofocus for quantitative volumetric microscopy of whole mouse brains / 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.; Pavone, F. S.. - In: NATURE METHODS. - ISSN 1548-7091. - ELETTRONICO. - 18:(2021), pp. 953-958. [10.1038/s41592-021-01208-1]
Universal autofocus for quantitative volumetric microscopy of whole mouse brains
Silvestri, L.
;Costantini, I.;Di Giovanna, A. P.;Mazzamuto, G.;Franceschini, A.;Checcucci, C.;Frasconi, P.;Pavone, F. S.
2021
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.File | Dimensione | Formato | |
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