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Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes

Nature Biotechnology volume 40, pages 1654–1662 (2022)Cite this article

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Abstract

Cells in complex organisms undergo frequent functional changes, but few methods allow comprehensive longitudinal profiling of living cells. Here we introduce scission-accelerated fluorophore exchange (SAFE), a method for multiplexed temporospatial imaging of living cells with immunofluorescence. SAFE uses a rapid bioorthogonal click chemistry to remove immunofluorescent signals from the surface of labeled cells, cycling the nanomolar-concentration reagents in seconds and enabling multiple rounds of staining of the same samples. It is non-toxic and functional in both dispersed cells and intact living tissues. We demonstrate multiparameter (n ≥ 14), non-disruptive imaging of murine peripheral blood mononuclear and bone marrow cells to profile cellular differentiation. We also show longitudinal multiplexed imaging of bone marrow progenitor cells as they develop into neutrophils over 6 days and real-time multiplexed cycling of living mouse hepatic tissues. We anticipate that SAFE will find broad utility for investigating physiologic dynamics in living systems.

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Fig. 1: Multiplexed temporospatial profiling in living cells.
Fig. 2: Mechanisms, synthesis and kinetics.
Fig. 3: Rapid, durable and non-toxic multiplexing of living cells.
Fig. 4: SAFE imaging of living hepatic tissue.
Fig. 5: SAFE imaging of living bone marrow.
Fig. 6: Longitudinal profiling of neutrophil differentiation.

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Data availability

All data that support the observations and conclusions of the study are included in the manuscript and its Supplementary Information. Raw multi-channel and/or z-stack source data from time series images are available in TIF format at https://doi.org/10.5281/zenodo.6482316.

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Acknowledgements

We are grateful to the Sykes Laboratory for preparing live bone marrow cells and to C. Carlson-O’Fallon for assistance with imaging data analysis. This work was supported, in part, by grants from the CSB development fund (J.C.T.C.), R01CA257623 (R.W.), UH3CA202637 (R.W.), R01CA206890 (R.W. and M.P.), P01CA069246 (R.W.), U01CA206997 (R.W.), P01CA240239 (M.P.) and R01CA229777 (R.W.). J.K. was supported by the Schmidt Science Fellows and K99CA256353.

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Authors and Affiliations

  1. Center for Systems Biology, Massachusetts General Hospital, Boston, MA, USA

    Jina Ko, Juhyun Oh, Rainer H. Kohler, Evangelia Bolli, Mikael J. Pittet, Claudio Vinegoni, Ralph Weissleder & Jonathan C. T. Carlson

  2. Institute of Applied Synthetic Chemistry, TU Wien, Vienna, Austria

    Martin Wilkovitsch & Hannes Mikula

  3. Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland

    Evangelia Bolli & Mikael J. Pittet

  4. Ludwig Institute for Cancer Research, Lausanne Branch, Zurich, Switzerland

    Mikael J. Pittet

  5. AGORA Cancer Center, Lausanne, Switzerland

    Mikael J. Pittet

  6. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA

    David B. Sykes

  7. Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    David B. Sykes & Jonathan C. T. Carlson

  8. Department of Systems Biology, Harvard Medical School, Boston, MA, USA

    Ralph Weissleder

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Contributions

Design: J.C.T.C., J.K. and R.W. Synthesis: M.W., H.M. and J.C.T.C. Experiments: J.K., J.O., E.B. and J.C.T.C. Data analysis: all authors. Writing: J.C.T.C., J.K., R.W. and all others.

Corresponding authors

Correspondence to Ralph Weissleder or Jonathan C. T. Carlson.

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Competing interests

The authors declare the following competing interests. J.C.T.C., R.W. and H.M. declare the filing of a patent (PCT/US2021/053439, pending; Bioorthogonal linkers and reactions), which was assigned to Massachusetts General Hospital. R.W. is a consultant to ModeRNA, Tarveda Therapeutics, Lumicell, Seer, Earli, Aikili Biosystems and Accure Health, consultancies that are unrelated to the subject matter of this work.

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Nature Biotechnology thanks Christian Schürch and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Information

Supplementary Tables 1 and 2, Supplementary Figs. 1–11, Synthetic Methods and chemical characterization data

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Supplementary Video 1

Time lapse imaging of SAFE cycling in living hepatic tissue.

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Ko, J., Wilkovitsch, M., Oh, J. et al. Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes. Nat Biotechnol 40, 1654–1662 (2022). https://doi.org/10.1038/s41587-022-01339-6

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