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Intrinsic fluorophores and their associated biological processes exhibit dynamics on a wide range of timescales throughout the heterogeneous milieu of living cells. Conventional time-lapse autofluorescence intensity imaging is best suited for monitoring slow physiological functions such as changes in cellular morphology, cell migration, and intracellular distribution that take place on a timescale of seconds to minutes. In contrast, ultrafast (10− 12–10− 7 s) time-resolved fluorescence measurements can probe molecular dynamics, such as excited-state processes and rotational dynamics, which are acutely sensitive to the chemical structure, intermolecular interactions, and microenvironment of a given fluorophore. Time-resolved fluorescence and anisotropy measurements are uniquely suited for a detailed