Claudio Vinegoni, Ph.D. | Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes

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

Ko, J. A., Wilkovitsch, M., Oh, J., Kohler, R. H., Bolli, E., Pittet, M. J., Vinegoni, C., Sykes, D. B., Mikula, H., Weissleder^#, R., and Carlson^#, J. C. T.

Nature Biotechnology 2022

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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. Live cells and tissues are imaged over long time periods using a fast, non-toxic click chemistry.
@article{2022-NB, author= {Ko, J. A. and Wilkovitsch, M. and Oh, J. and Kohler, R. H. and Bolli, E. and Pittet, M. J. and Vinegoni, C. and Sykes, D. B. and Mikula, H. and Weissleder<sup>#</sup>, R. and Carlson<sup>#</sup>, J. C. T.}, title= {Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes}, journal= {Nature Biotechnology}, alternatejournal = {Nat. Biotechnol.}, year = {1998}, pages = {16}, issn = {1087-0156}, doi = {10.1038/s41587-022-01339-6}, pmid= {35654978} }
35654978

10.1038/s41587-022-01339-6

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. Live cells and tissues are imaged over long time periods using a fast, non-toxic click chemistry."

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Ko, J. A., Wilkovitsch, M., Oh, J., Kohler, R. H., Bolli, E., Pittet, M. J., Vinegoni, C., Sykes, D. B., Mikula, H., Weissleder^#, R., & Carlson^#, J. C. T. (2022). Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes. Nature Biotechnology, 16. https://doi.org/10.1038/s41587-022-01339-6