Fluorescence Lifetime Imaging Microscopy (FLIM) measures the time a fluorophore spends in its excited state rather than the intensity of emitted light. Because fluorescence lifetime is sensitive to the local molecular environment — including proximity to quenchers, changes in conformation, and binding state — it provides a read-out of phosphorylation status at the level of individual living cells.
We develop peptide-based FLIM biosensors in which phosphorylation drives an intramolecular rearrangement that alters the lifetime of an attached fluorophore. Delivered into living cells via cell-penetrating peptide sequences, these probes report kinase activity with subcellular spatial resolution and in real time, without the need for genetic encoding.
This approach enables us to map heterogeneity in kinase signaling across cell populations, track dynamic changes in response to drug treatment, and study compartment-specific regulation — capabilities not accessible through bulk biochemical assays.