The biochemical activities underlying cell-fate decisions vary profoundly even in genetically identical cells. But such non-genetic heterogeneity remains refractory to current imaging methods because their capacity to monitor multiple biochemical activities in single living cells over time remains limited. Recently we deployed a family of newly designed GFP-like sensors (NyxBits) with fast photon-counting electronics and bespoke analytics (NyxSense) in multiplexed biochemical imaging to define a network determining the fate of single cells exposed to the DNA-damaging drug cisplatin. By simultaneously imaging a tri-nodal network comprising the cell-death proteases Caspase-2, -3, and -9, we reveal unrecognized single-cell heterogeneities in the dynamics and amplitude of caspase activation that signify survival versus cell death via necrosis or apoptosis.

Thus, multiplexed biochemical imaging reveals cellular populations and biochemical states, invisible to other methods, underlying therapeutic responses to an anticancer drug. Our work develops widely applicable tools to monitor the dynamic activation of biochemical networks at single-cell resolution. It highlights the necessity to resolve patterns of network activation in single cells, rather than the average state of individual nodes, to define, and potentially control, mechanisms underlying cellular decisions in health and disease. Read more or here