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.

复杂生物体中的细胞经常发生功能变化，但很少有方法能够对活细胞进行全面的纵向分析。在这里，我们介绍了断裂加速荧光团交换 (SAFE)，这是一种使用免疫荧光对活细胞进行多重时空成像的方法。SAFE 使用快速生物正交点击化学从标记细胞表面去除免疫荧光信号，在几秒钟内循环纳摩尔浓度的试剂，并对同一样本进行多轮染色。它在分散的细胞和完整的活组织中都是无毒的和功能性的。我们展示了多参数 ( n ≥ 14)，对小鼠外周血单核细胞和骨髓细胞进行非破坏性成像以分析细胞分化。我们还展示了骨髓祖细胞在 6 天内发育成嗜中性粒细胞时的纵向多重成像，以及活体小鼠肝组织的实时多重循环。我们预计 SAFE 将在研究生命系统的生理动力学方面找到广泛的用途。