Many cancers exhibit deregulated activity of protein kinase enzymes, but not all are sensitive to inhibitor drugs, largely because phosphorylation dynamics in complex tissues are not well understood.[1] Live, subcellular analysis can reveal the details of kinase signaling in mixed populations of cells.[2] Current tools to image kinase activity in situ depend on intensity-based measurements (such as fluorescence and Forster resonance energy transfer) that can be limited by spectral bleed-through and photobleaching.[3] We report a cell-penetrating peptide biosensor for dynamic monitoring of phosphorylation by Abl kinase based on fluorescence lifetime imaging microscopy (FLIM).[4] FLIM, which is not confounded by photobleaching or cellular autofluorescence, was applied to detect phosphorylation-dependent fluorophore lifetime shifts (1–2 ns) in intact, living cells (Fig. 1). We established the dependence of the fluorophore lifetime shift on phosphorylation specifically by Abl kinase, mapped the fluorophore intensity and lifetime components to quantify subcellular phosphorylation, and monitored kinase inhibition in real time. This approach should be generalizable to other kinases and provides a new method for interrogating real-time, subcellular signaling activities in cell populations that are not amenable to expression of genetically engineered biosensor proteins. Figure 1 FLIM to detect phosphorylation-dependent fluorophore lifetime shifts for biosensors in intact, live cells Measuring subcellular kinase activity in living cells remains a major challenge. Genetically encoded Forster resonance energy transfer (FRET) biosensors can be used for this purpose in simple cell-based assays and basic research applications.[3, 5] These sensors take advantage of binding between phosphorylated sequences and phosphopeptide binding domains to bring two fluorescent proteins close enough for energy transfer to occur. However, expressing genetically engineered proteins in cells has challenges, including a) uniform transfection and expression of protein fluorophores (a roadblock for applications in primary patient-derived cells or tissues) and b) the large labels which can affect substrate function and interaction with a kinase.[5–6] Small molecule fluorophores are able in principle to be less disruptive to function, and many are available for which excitation and emission do not overlap with expressible fluorophores (enabling multiplexed co-localization experiments).[7] These have been applied to detect phosphorylation in cells via fluorescence intensity increases.[8] Low signal to noise is a limitation of FRET, and intensity-based fluorescence is confounded by photobleaching when experiments are conducted over long time periods, making it difficult to interpret subcellular fluctuations at high spatial and temporal resolution.[6] FLIM is not affected by photobleaching or intensity and has the potential for single molecule monitoring.[4, 9] Also, time-correlated single photon counting FLIM is capable of highly resolved discrimination between species exhibiting very small differences in lifetimes (even sub-nanosecond), facilitating the mapping of exquisite detail in subcellular images. Here we describe the first demonstration of a FLIM-based phosphorylation biosensor technology that has the potential to circumvent key technological gaps as a new strategy for studying intracellular signaling biology. We combined the delivery of organic fluorophore-tagged kinase substrate peptide probes with time-resolved FLIM to visualize the details of kinase activity in live, intact cells (Fig. 1). The biosensor consists of an Abl substrate peptide containing the “Abltide” substrate sequence[10] tagged with a Cy5 fluorophore and a cell penetrating peptide (Abl-TAT: GGEAIYAAPCCy5GGRKKRRQRRRPQ) (Fig. 2).[11] The substrate portion (bold) is relatively selective for the c-Abl kinase (Abl1) over other tyrosine kinases, however it is phosphorylated by the Abl family member named Abl-related gene (Arg, also known as Abl2).[12] Abl1 and Abl2 are highly homologous and share many functions in normal cells.[13] In this work, “Abl kinase” denotes both Abl1 and Abl2. We used FLIM instrumentation with picosecond pulsing lasers[9a, 9b] to measure Cy5 lifetime for the unphosphorylated biosensor and a phosphorylated derivative in solution and in live cells. Figure 2 Peptide-based Abl kinase biosensor In solution, lifetime differences between the phosphorylated and unphosphorylated Abl-TAT peptide species were not significant (see supporting information, Fig. S1a), indicating that phosphorylation alone is not sufficient to elicit a change in the rate of fluorescence signal decay for the Abl-TAT peptide sensor. However, in the presence of c-Abl kinase at 1:1 ratio, robust lifetime differences were observed, and this phenomenon was blocked by pre-incubation of the kinase with higher ratios of unlabelled phosphopeptide (Fig. S1b). This effect likely arises from the more drastic change in the local environment of the fluorophore that could occur upon binding of the phosphopeptide with the protein, probably through the kinase SH2 domain.[14] Since the physiochemical basis for the lifetime shift was still somewhat unknown, standards were established in NIH3T3 immortalized mouse embryonic fibroblast cells (MEFs)[15] to assess phosphorylation- and Abl kinase-dependence of the lifetime shifts by using three key negative controls (which exhibited lower lifetimes in cells): Cy5 alone, a non-phosphorylatable Y→F peptide sensor analog (Abl-F-mutant) (both in control MEFs expressing Abl kinase) and the Abl-TAT biosensor in Abl(−/−) knockout cells[15] (Fig. 3B, C and E and Fig. S2). Average lifetimes per cell for multiple cells were calculated and plotted to show the distribution of lifetimes observed for the biosensor and each control (Fig. 3G). The distributions were determined to be non-Gaussian, so non-parametric ANOVA with a Dunn’s post-test (described in the Methods section) was used to evaluate statistical significance (P