Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M

Nat Protoc. 2020 Mar;15(3):840-876. doi: 10.1038/s41596-019-0269-9. Epub 2020 Jan 22.


Simultaneous observation of 3D chromatin organization and transcription at the single-cell level and with high spatial resolution may hold the key to unveiling the mechanisms regulating embryonic development, cell differentiation and even disease. We recently developed Hi-M, a technology that enables the sequential labeling, 3D imaging and localization of multiple genomic DNA loci, together with RNA expression, in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe rehybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos in microfluidic chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4-5 d, including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1-2 d to complete all rounds of labeling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells or organization of chromatin within complex tissues.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatin
  • Chromosomes*
  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism
  • Drosophila / embryology
  • Fluorescent Dyes
  • Gene Expression Regulation, Developmental / physiology*
  • Image Processing, Computer-Assisted*
  • In Situ Hybridization, Fluorescence / methods
  • RNA / chemistry
  • RNA / genetics
  • RNA / metabolism
  • Transcription, Genetic / physiology*


  • Chromatin
  • Fluorescent Dyes
  • RNA
  • DNA