Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy
Authors: Silvestri L., Bria A., Costantini I., Sacconi L., Peng H., Iannello G., Pavone F.S.
Autors Affiliation: European Laboratory for Non-linear Spectroscopy (LENS); Integrated Research Centre, University Campus Bio-medico of Rome; DAEMI, University of Cassino; National Institute of Optics (CNR-INO); Allen Institute for Brain Science; Department of Physics, University of Florence; ICON Foundation, Sesto Fiorentino, Italy
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.
Journal/Review: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume: 80 Pages from: e50696 to: e50696
KeyWords: Enhanced green fluorescent protein; Green fluorescent protein, anatomy and histology; Animal; Biosynthesis; Brain; Brain level; Brain mapping; Cerebellum; Chemistry; Confocal microscopy; Genetics; Mouse; Optical tomography; Procedures; Transgenic mouse, Animals; Brain; Brain Chemistry; Brain Mapping; Cerebellum; Green Fluorescent Proteins; Mice; Mice, Transgenic; Microscopy, Confocal; Tomography, OpticalDOI: 10.3791/50696Citations: 13data from “WEB OF SCIENCE” (of Thomson Reuters) are update at: 2020-01-12References taken from IsiWeb of Knowledge: (subscribers only)Connecting to view paper tab on IsiWeb: Click hereConnecting to view citations from IsiWeb: Click here