title: Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.
abstract: Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas - from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA UCLA-Caltech Medical Scientist Training Program, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, 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: 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: Maayan
authors.initials: M
authors.lastname: Schwarzkopf
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Ting-Fang
authors.initials: TF
authors.lastname: He
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Alon
authors.initials: A
authors.lastname: Greenbaum
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Chang Ho
authors.initials: CH
authors.lastname: Sohn
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Antti
authors.initials: A
authors.lastname: Lignell
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Harry M T
authors.initials: HM
authors.lastname: Choi
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA lcai@caltech.edu niles@caltech.edu viviana@caltech.edu.
authors.forename: Viviana
authors.initials: V
authors.lastname: Gradinaru
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA Division of Engineering & Applied Science, California Institute of Technology, Pasadena, CA 91125, USA lcai@caltech.edu niles@caltech.edu viviana@caltech.edu.
authors.forename: Niles A
authors.initials: NA
authors.lastname: Pierce
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA lcai@caltech.edu niles@caltech.edu viviana@caltech.edu.
authors.forename: Long
authors.identifier: 0000-0002-7154-5361
authors.initials: L
authors.lastname: Cai
mesh_terms: D000818:Animals; D004625:Embryo, Nonmammalian / Q000378:metabolism; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry*; D017404:In Situ Hybridization, Fluorescence; D012313:RNA / Q000235:genetics*; D015027:Zebrafish
chemical_list: D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D012313:RNA
keywords: Amplification; CLARITY; Single-molecule RNA