title: Micron-scale resolution optical tomography of entire mouse brains with confocal light sheet microscopy.
abstract: Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.
authors.affiliation: European Laboratory for Non-linear Spectroscopy (LENS).
authors.forename: Ludovico
authors.initials: L
authors.lastname: Silvestri
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bria
authors.forename: Irene
authors.initials: I
authors.lastname: Costantini
authors.forename: Leonardo
authors.initials: L
authors.lastname: Sacconi
authors.forename: Hanchuan
authors.initials: H
authors.lastname: Peng
authors.forename: Giulio
authors.initials: G
authors.lastname: Iannello
authors.forename: Francesco Saverio
authors.initials: FS
authors.lastname: Pavone
mesh_terms: D000818:Animals; D001921:Brain / Q000033:anatomy & histology*; D001923:Brain Chemistry; D001931:Brain Mapping / Q000379:methods*; D002531:Cerebellum / Q000033:anatomy & histology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal / Q000379:methods*; D041622:Tomography, Optical / Q000379:methods*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins