title: High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing.
abstract: Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, background-from off-target binding of FISH probes and cellular autofluorescence-can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues.
authors.affiliation: Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138.|Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Moffitt
authors.affiliation: Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138.|Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Junjie
authors.initials: J
authors.lastname: Hao
authors.affiliation: Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138.|Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Bambah-Mukku
authors.affiliation: Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138.|Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Tian
authors.initials: T
authors.lastname: Lu
authors.affiliation: Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138.|Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Catherine
authors.initials: C
authors.lastname: Dulac
authors.affiliation: Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138; zhuang@chemistry.harvard.edu.|Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.|Department of Physics, Harvard University, Cambridge, MA 02138.
authors.forename: Xiaowei
authors.identifier: 0000-0002-6034-7853
authors.initials: X
authors.lastname: Zhuang
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007031:Hypothalamus / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D015347:RNA Probes; D059010:Single-Cell Analysis / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D015347:RNA Probes
keywords: brain; fluorescence in situ hybridization; multiplexed imaging; single-cell transcriptomics; tissue clearing