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

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.

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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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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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 (2022). https://doi.org/10.1038/s41587-022-01339-6

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