title: Optimized Protocol for Imaging Cleared Neural Tissues Using Light Microscopy.
abstract: Understanding physical and chemical processes at an organismal scale is a fundamental goal in biology. While science is adept at explaining biological phenomena at both molecular and cellular levels, understanding how these processes translate to organismal functions remains a challenging problem. This issue is particularly significant for the nervous system where cell signaling and synaptic activities function in the context of broad neural networks. Recent progress in tissue clearing technologies lessens the barriers that previously prevented the study of large tissue samples while maintaining molecular and cellular resolution. While these new methods open vast opportunities and exciting new questions, the logistics of analyzing cellular processes in intact tissue have to be carefully considered. In this protocol, we outline a procedure to rapidly image intact brain tissue up to thousands of cubic millimeters. This experimental pipeline involves three steps: tissue clearing, tissue imaging, and data analysis. In an attempt to streamline the process for researchers entering this field, we address important considerations for each of these stages and describe an integrated solution to image intact biological tissues. Hopefully, this optimized protocol will lower the barrier of implementing high-resolution tissue imaging and facilitate the investigations of mesoscale questions at molecular and cellular resolution.
authors.affiliation: Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.|Sainsbury Wellcome Centre for Neural Circuits and Behaviour, University College London, London, UK.
authors.forename: Yoh
authors.initials: Y
authors.lastname: Isogai
authors.affiliation: Harvard Center for Biological Imaging, Harvard University, Cambridge, MA, USA.
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Richardson
authors.affiliation: Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
authors.forename: Catherine
authors.initials: C
authors.lastname: Dulac
authors.affiliation: Department of Psychological and Brain Sciences, University of Massachusetts, 230 Stockbridge Road, Amherst, MA, 01003-9316, USA. jbergan@umass.edu.
authors.forename: Joseph
authors.initials: J
authors.lastname: Bergan
mesh_terms: D000818:Animals; D015415:Biomarkers; D001921:Brain / Q000000981:diagnostic imaging / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional / Q000379:methods; D016247:Information Storage and Retrieval; D051379:Mice; D008853:Microscopy / Q000379:methods*; D009417:Nerve Tissue / Q000166:cytology* / Q000378:metabolism*; D059906:Neuroimaging / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D012984:Software
chemical_list: D015415:Biomarkers
keywords: CLARITY; Confocal; Lightsheet; OptiView; Tissue clearing; Whole brain imaging