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


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.

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Fig. 1: Outline of the Hi-M protocol.
Fig. 2: Design of Oligopaints.
Fig. 3: Oligo pool amplification.
Fig. 4: Example results.
Fig. 5: Procedure for attaching embryos to the microfluidic chamber.
Fig. 6: Scheme of hybridization/imaging cycles.
Fig. 7: Hi-M setup.
Fig. 8: Hi-M image acquisition flowchart.
Fig. 9: Hi-M image analysis flowchart.
Fig. 10: Data analysis.
Fig. 11: Hi-M output.

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Data and code availability

The code used and described in this paper and the experimental dataset used to construct Fig. 11 have been uploaded to Additional advice on how to use these can be obtained from the authors upon reasonable request. Further information and requests for resources, reagents and software should be directed to and will be fulfilled by the lead contact, M.N. (


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This project received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Program (grant 724429). This work also benefited from support from Labex EpiGenMed, an ‘Investments for the Future’ program (grant ANR-10-LABX-12-01). We acknowledge the France-BioImaging infrastructure supported by the French National Research Agency (grant ANR-10-INBS-04, ‘Investments for the Future’).

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Authors and Affiliations



A.M.C.G. and M.N. designed the experiments. A.M.C.G. and M.N. designed the Oligopaints probes. C.H. amplified and purified the Oligopaints libraries. A.M.C.G., C.H., S.M.E. and J.G. developed the RNA/DNA staining protocol; A.M.C.G., S.M.E., J.G. and D.I.C. conducted the experiments. J.-B.F. designed and built the microscopy setup and acquisition software. M.N. developed the software for image analysis. A.M.C.G., and J.G. analyzed the data. A.M.C.G., D.I.C. and M.N. wrote the manuscript. All authors reviewed and commented on the manuscript.

Corresponding author

Correspondence to Marcelo Nollmann.

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Cardozo Gizzi, A. M. et al. Mol. Cell 74, 212–222.e5 (2019):

Integrated supplementary information

Supplementary Figure 1 Microfluidic setup plan.

Schematic representation of microfluidics used in Hi-M setup. Buffers and readout probes are selected using a combination of three eight-way HVXM8-5 valves connected in series. The fluid handling circuit consist of 21 tubes connected each to one of the valves. Hi-M wash buffer and 2× SSC pass through an online degassing unit before connecting to the corresponding valve. The other 19 valves are connected to 17 readout solution tubes, the chemical bleaching buffer and the acquisition solution. The sample is mounted to the FCS2 flow chamber. Continuous flow is created by a negative-pressure pump. Before starting the experiment, the system is fill with 2× SCC buffer, assuring the removal of all air bubbles. Microfluidics is automatically controlled using a homemade LabView 2015 software. Image adapted with permission from ref. 3, Elsevier.

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Supplementary Information

Supplementary Fig. 1 and Supplementary Tables 2–4.

Reporting Summary

Supplementary Table 1

Oligo pool sequences

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Cardozo Gizzi, A.M., Espinola, S.M., Gurgo, J. et al. Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M. Nat Protoc 15, 840–876 (2020).

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