A modular scaffold for cellularly-retained fluorogenic probes for sensitive cell-resolved bioactivity imaging

Here, we develop a general design for high-quality fluorogenic activity probes to quantify biochemical processes within live cells, via the release of a fully cell-retained, bright fluorescent soluble product upon reaction. Live cell probes must be membrane-permeable to access intracellular biochemistry, but often that means their fluorophore products are similarly permeable resulting in rapid signal loss from the activating cell, which limits their cell-by-cell resolution as well as their sensitivity for quantifying low-turnover processes. Current strategies to retain fluorescent products within cells usually disrupt native biology e.g. by non-specific alkylation or solid precipitation. Here, scanning charge- and polarity-based approaches to swap from permeable to cell-retained states, we developed a bright fluorogenic rhodol-based platform, Trappable Green (TraG), balancing all key requirements for signal integration (rapid probe entry, but effective product retention, across many cell lines) and being modular so it can be adapted to quantify many biochemical target types (examples shown here include probes for GSH, TrxR, and H₂O₂). The simple and rugged TraG scaffold can now permit straightforward implementation in a range of cell-retained enzyme activity probes, which will enable more accurate cell-resolved imaging as well as higher-sensitivity integration of low-turnover processes, without the drawbacks of alkylation or precipitation-based strategies.