Highlights d Large numbers of fluorescent biosensors can be concurrently tracked in barcoded cells d Biosensor activities are synchronized in mixed populations of barcoded cells d Deep learning models facilitate image analysis for biosensor barcoding d Simultaneous biosensor tracking reveals signaling network structures and interactions

Fluorescent biosensors allow for real-time monitoring of biochemical activities in cells, but their multiplexing capacity is severely limited by the availability of spectral space. We overcome this problem by developing a set of barcoding proteins that are spectrally separable from commonly used FRET (fluorescence resonance energy transfer)-based and single-fluorophore biosensors. Mixed populations of barcoded cells expressing different biosensors can be concurrently imaged and computationally unmixed to achieve highly multiplexed tracking of biochemical activities in live cells.

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