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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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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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.
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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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