We want to provide a reference atlas of the spatial distribution of different neuronal types across the entire mouse brain. To this aim, we use transgenic animals where selected neuronal populations are fluorescently labeled. Intact brains cleared with CLARITY-TDE are imaged with a custom-made light sheet microscope.
The same clearing and imaging pipeline is applied to a transgenic mouse strain where fluorescent proteins are expressed under the promoter of the immediate early gene c-fos (TRAP mice, Guenthner et al. 2013). In this way, we can produce a brain-wide cell-resolution map of the neuronal activation patterns related to specific behaviours.
We are constantly working to improve the quality and resolution of light-sheet images. To this aim, we coupled confocal line detection to light sheet illumination. Recently, we also developed an image-based real-time autofocus system (RAPID, see below) to maintain image quality high throughout the sample, and implemented Bessel-beam illumination to reduce shadowing artifacts.
Ludovico Silvestri, Caroline Muellenbroich, Irene Costantini, Giuseppe Sancataldo, Antonino Paolo Di Giovanna, Vladislav Gavryusev, Giuseppe De Vito, Giacomo Mazzamuto, Leonardo Sacconi, Francesco Saverio Pavone.
Giulio Iannello, Facoltà di Ingegneria, Università Campus Bio-Medico di Roma
Paolo Frasconi, Dipartimento di Ingegneria dell’Informazione - Università di Firenze
Jan G. Bjaalie, Head of Neuroinformatics division, Institute of Basic Medical Sciences, University of Oslo, Norway
Trygve Leergaard, Professor of Neural Systems, Institute of Basic Medical Sciences, University of Oslo, Norway
Hanchuan Peng, Hanchuan Peng, Associate Investigator at Allen Brain Institute, Seattle, US
Bruno Weber, Professor of Multimodal Experimental Imaging, Universitaet Zuerich, Switzerland
Selected Recent Publications
Ludovico Silvestri, Marie Caroline Muellenbroich, Irene Costantini, Antonino Paolo Di Giovanna, Leonardo Sacconi, Francesco Saverio Pavone, (2017) RAPID: Real-time image-based autofocus for all wide-field optical microscopy systems, bioRxiv 170555 (pre-print version) doi: https://doi.org/10.1101/170555
Allegra Mascaro, A.L.; Silvestri, L.; Sacconi, L.; Pavone, F.S. (2015) Towards a comprehensive understanding of brain machinery by correlative microscopy. J Biomed Opt. Jun;20(6):61105
Allegra Mascaro, A.L., Costantini, I., Margoni, E., Iannello, G., Bria, A., Sacconi, L., Pavone, F.S. (2015) Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging. Biom. Optics Express, 6(11):4483-4492
Costantini, I., Ghobril, J.P., Di Giovanna, A.P., Allegra Mascaro, A.L., Silvestri, L., Müllenbroich, M.C., Onofri, L., Conti, V., Vanzi, F., Sacconi, L., Guerrini, R., Markram, H., Iannello, G., Pavone F.S. (2015) A versatile clearing agent for multi-modal brain imaging. Sci Rep. 7(5):9808
M. Caroline Müllenbroich, Ludovico Silvestri, Leonardo Onofri, Irene Costantini, Marcel van’t Hoff, Leonardo Sacconi, Giulio Iannello, Francesco S. Pavone, “Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains,” Neurophotonics 2(4), 041404 (29 June 2015). http://dx.doi.org/10.1117/1.NPh.2.4.041404
Silvestri, L.; Allegra Mascaro, A.L.; Costantini, I., Sacconi, L.; Pavone, F.S. (2014) Correlative two-photon and light sheet microscopy. Methods 66(2):268-272
L. Silvestri, L. Sacconi, F. S. Pavone, Correcting spherical aberrations in confocal light sheet microscopy: A theoretical study, Microscopy Research and Technique, Vol. 77, Issue 7, pp. 483‑491, DOI: 10.1002/jemt.22330, 2014, January 3
System and method of measuring the focusing of an optical instrument : Italian Patent pending n. 102016000132604 (“Sistema e metodo di misura della focalizzazione di uno strumento ottico” -) of 30/12/2016. Applicants: LENS (40%), UNIFI (30%) e CNR (30%). Inventors: L. Silvestri, M.C. Muellenbroich, L. Sacconi e F.S. Pavone.