We initially collaborated with Joseph Irudayaraj (UIUC) and are now funded separately by the NCI IMAT program (grant #R33CA217780) to develop a fluorescence lifetime imaging strategy for peptide kinase biosensors. In this work, we found that upon phosphorylation of a fluorophore-tagged Abltide substrate peptide, the fluorescence lifetime would increase. Through control experiments, we attributed this to peptide-protein interaction between the phosphorylated product peptide and the kinase (or other nearby proteins), most likely through phosphotyrosine peptide-binding SH2 domains. We have used this strategy to image the phosphorylation dynamics of the Abl substrate in live cells in the absence and presence of the Abl inhibitor imatinib. In the current IMAT-funded project, we are characterizing the SH2 binding and developing multiplexed analysis