Bio-orthogonal chemistries are constructed around chemical partners that remain functional and react selectively even in the complex biological milieu. The team at MGH CSB and our collaborators have been at the forefront of exploring these reactions, developing the singularly fast and adaptable bio-orthogonal ligation of tetrazines (Tz) and trans-cyclooctenes (TCO) into a versatile toolkit for connecting and disconnecting biomolecules on demand. Leveraging these efforts, we aimed to re-tool serial immunostaining to make it compatible with living cells and tissues: fast, nontoxic, and high-performance. We embedded a highly stable and efficient scissile linker (C2TCO, invented at CSB) within a scaffold that exploits non-covalent fluorophore-quencher interactions to turbocharge its reactivity. Paired with an optimized “Tz scissors”, this architecture for scission-accelerated fluorophore exchange (SAFE) boosts the intrinsic Tz/TCO reaction rate by more than 3,000-fold, enabling us to erase fluorescent signals (>99% clearance) from live antibody-labelled cells in seconds at nontoxic nanomolar concentrations.
The chemical machinery of SAFE performs cleanly within living cells and tissues: SAFE-labelled antibodies in four color channels can generate multiplexed profiles of living mouse peripheral blood mononuclear cells in microwell suspension, bone marrow cells in adherent culture, and sections of freshly explanted hepatic tissue in ≥ 14 channels/colors. Serial images recorded these living cells progressing through mitosis amidst the scission process and differentiating across six days, while confocal time-lapse imaging of living tissues captured cellular migration in rich molecular detail.
SAFE staining can readily define cellular subsets within complex populations/architectures and over extended timeframes—creating the first method for multiplexed temporospatial profiling of living cells and tissues in their biological context.