title: Optimization of CLARITY for Clearing Whole-Brain and Other Intact Organs
abstract: The development, refinement, and use of techniques that allow high-throughput imaging of whole brains with cellular resolution will help us understand the complex functions of the brain. Such techniques are crucial for the analysis of complete neuronal morphology-anatomical and functional-connectivity, and repeated molecular phenotyping. CLARITY is a recently introduced technique that produces structurally intact, yet optically transparent tissue, which may be labeled and imaged without sectioning. However, the utility of this technique depends on several procedural variables during the process in which the light-scattering lipids in a tissue are replaced by a transparent hydrogel matrix. Here, we systematically varied a number of factors (including temperature, hydrogel composition, and polymerization conditions) to provide an optimized, highly replicable CLARITY procedure for clearing mouse brains. We found that for these preparations optimal tissue clearing requires electrophoresis (and cannot be achieved with passive clearing alone) for 5 d with a combination of 37 and 55°C temperature. Although this protocol is optimized for brains, we also show that it can be used to clear and analyze a variety of organs. Brain or other tissue prepared using this protocol is suitable for high-throughput imaging with confocal or single-plane illumination microscopy.
authors.affiliation: Program in Neurosciences and Mental Health, Hospital for Sick Children , Toronto, Ontario M5G 1X8, Canada ; Institute of Medical Sciences, University of Toronto , Toronto, Ontario M5G 1X8, Canada ; Departments of Psychology and Physiology , University of Toronto , Toronto, Ontario M5G 1X8, Canada.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Epp
authors.affiliation: Program in Neurosciences and Mental Health, Hospital for Sick Children , Toronto, Ontario M5G 1X8, Canada ; Institute of Medical Sciences, University of Toronto , Toronto, Ontario M5G 1X8, Canada ; Departments of Psychology and Physiology , University of Toronto , Toronto, Ontario M5G 1X8, Canada.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Niibori
authors.affiliation: Program in Neurosciences and Mental Health, Hospital for Sick Children , Toronto, Ontario M5G 1X8, Canada ; Institute of Medical Sciences, University of Toronto , Toronto, Ontario M5G 1X8, Canada ; Departments of Psychology and Physiology , University of Toronto , Toronto, Ontario M5G 1X8, Canada.
authors.forename: Hwa-Lin
authors.initials: HL
authors.lastname: Liz Hsiang
authors.affiliation: Program in Neurosciences and Mental Health, Hospital for Sick Children , Toronto, Ontario M5G 1X8, Canada ; Institute of Medical Sciences, University of Toronto , Toronto, Ontario M5G 1X8, Canada ; Departments of Psychology and Physiology , University of Toronto , Toronto, Ontario M5G 1X8, Canada.
authors.forename: Valentina
authors.initials: V
authors.lastname: Mercaldo
authors.affiliation: Department of Bioengineering and Psychiatry, Stanford University , Stanford, California 94305.
authors.forename: Karl
authors.initials: K
authors.lastname: Deisseroth
authors.affiliation: Program in Neurosciences and Mental Health, Hospital for Sick Children , Toronto, Ontario M5G 1X8, Canada ; Institute of Medical Sciences, University of Toronto , Toronto, Ontario M5G 1X8, Canada ; Departments of Psychology and Physiology , University of Toronto , Toronto, Ontario M5G 1X8, Canada.
authors.forename: Sheena A
authors.initials: SA
authors.lastname: Josselyn
authors.affiliation: Program in Neurosciences and Mental Health, Hospital for Sick Children , Toronto, Ontario M5G 1X8, Canada ; Institute of Medical Sciences, University of Toronto , Toronto, Ontario M5G 1X8, Canada ; Departments of Psychology and Physiology , University of Toronto , Toronto, Ontario M5G 1X8, Canada.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Frankland
keywords: 3D imaging; CLARITY; light sheet microscopy; neuron morphology; tissue clearing; whole-brain imaging