title: Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains.
abstract: In order to observe and quantify long-range neuronal connections in intact mouse brain by light microscopy, it is first necessary to clear the brain, thus suppressing refractive-index variations. Here we describe a method that clears the brain and preserves the signal from proteinaceous fluorophores using a pH-adjusted non-aqueous index-matching medium. Successful clearing is enabled through the use of either 1-propanol or tert-butanol during dehydration whilst maintaining a basic pH. We show that high-resolution fluorescence imaging of entire, structurally intact juvenile and adult mouse brains is possible at subcellular resolution, even following many months in clearing solution. We also show that axonal long-range projections that are EGFP-labelled by modified Rabies virus can be imaged throughout the brain using a purpose-built light-sheet fluorescence microscope. To demonstrate the viability of the technique, we determined a detailed map of the monosynaptic projections onto a target cell population in the lateral entorhinal cortex. This example demonstrates that our method permits the quantification of whole-brain connectivity patterns at the subcellular level in the uncut brain.
authors.affiliation: Max Planck Institute for Medical Research, Heidelberg, Germany.
authors.forename: Martin K
authors.initials: MK
authors.lastname: Schwarz
authors.affiliation: Max Planck Institute for Medical Research, Heidelberg, Germany.
authors.forename: Annemarie
authors.initials: A
authors.lastname: Scherbarth
authors.affiliation: Max Planck Institute for Medical Research, Heidelberg, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Sprengel
authors.affiliation: German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Johann
authors.initials: J
authors.lastname: Engelhardt
authors.affiliation: Center for Molecular Biology (ZMBH), University of Heidelberg, Heidelberg, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Theer
authors.affiliation: Max Planck Institute for Medical Research, Heidelberg, Germany.
authors.forename: Guenter
authors.initials: G
authors.lastname: Giese
mesh_terms: D000433:1-Propanol / Q000737:chemistry; D000818:Animals; D001921:Brain / Q000166:cytology* / Q000821:virology; D001931:Brain Mapping / Q000379:methods*; D002460:Cell Line; D006224:Cricetinae; D049452:Green Fluorescent Proteins; D021621:Imaging, Three-Dimensional / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence / Q000379:methods*; D009474:Neurons / Q000166:cytology* / Q000821:virology; D011820:Rabies virus; D020002:tert-Butyl Alcohol / Q000737:chemistry
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000433:1-Propanol; D020002:tert-Butyl Alcohol