Expanding the multiplexing capability of HIDE probes via fluorescence lifetime imaging microscopy

Fluorescence lifetime imaging microscopy (FLIM) measures the average time that a fluorophore spends in the excited state. FLIM is exceptionally useful as an imaging tool because the lifetime of a fluorophore is independent of its excitation and emission profiles. As such, even spectrally identical fluorophores can often be distinguished on the basis of their fluorescence lifetimes. Furthermore, given the dependence of fluorescence lifetime on the relative magnitude of radiative and nonradiative decay rates, it is possible to both predict and modulate the lifetime of a single fluorophore for applications in FLIM multiplexing. Herein, we provide an overview of the protocols necessary for transfection-free FLIM multiplexing in live cells. Protocols are provided for the in vitro reactions required to initially screen bioorthogonal handles and dyes, incubation of the small molecule probes required for FLIM multiplexing with live mammalian cells, and the setup of FLIM experiments. We also provide a discussion of methods available to analyze FLIM data to extract lifetime data.