title: Single-cell phenotyping within transparent intact tissue through whole-body clearing.
abstract: Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Treweek
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA; Division of Dermatology, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
authors.forename: Rajan P
authors.initials: RP
authors.lastname: Kulkarni
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Deverman
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Chun-Kan
authors.initials: CK
authors.lastname: Chen
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Lubeck
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Sheel
authors.initials: S
authors.lastname: Shah
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Long
authors.initials: L
authors.lastname: Cai
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA. Electronic address: viviana@caltech.edu.
authors.forename: Viviana
authors.initials: V
authors.lastname: Gradinaru
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology; D002477:Cells / Q000145:classification* / Q000378:metabolism; D005453:Fluorescence; D021621:Imaging, Three-Dimensional / Q000379:methods*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008855:Microscopy, Electron, Scanning; D010641:Phenotype; D059010:Single-Cell Analysis; D051598:Whole Body Imaging
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