Fluorescence Lifetime Imaging in Plants: Practical guidelines for multiplexing, label-free imaging and data analysis

Fluorescence Lifetime Imaging Microscopy (FLIM) is becoming a key technique for live-cell multiplexing and label-free detection of endogenous fluorescence in animal systems. Its potential in plant biology, however remains largely unexploited, despite its integration into a number of commercial microscopy setups. Here, we build a systematic, subcellular FLIM reference library for a panel of genetically-encoded fluorophores. Lifetime imaging of different fluorescent reporters targeted to distinct organelles (nucleus, plasma membrane, endoplasmic reticulum, etc.) and subsequent analysis of the decay curves using different modes allowed us to simultaneously discriminate up to four spectrally overlapping fluorophores solely by lifetime differences in specific subcellular compartments. Remarkably, fluorophores with lifetimes differing by as little as 0.1 ns can be reliably discriminated using one of these modes, namely Phasor-based analysis. Moreover, we show that the same fluorophores exhibit compartment-specific lifetime shifts, enabling Phasor separation of identical tags residing in different organelles. Finally, we extended the Phasor approach to label-free imaging of endogenous plant fluorescence. Together, these results establish FLIM-Phasor as a versatile, multiplex-capable tool for plant cell biology, opening new avenues for imaging strategies that yield higher content information at both cellular and tissue-level resolution.