Imaging Vacuolar Anthocyanins with Fluorescence Lifetime Microscopy (FLIM).

Anthocyanins are intrinsically fluorescent pigments that accumulate in plant vacuoles. We have developed a platform to analyze the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), under in vitro and in vivo conditions. Fluorescence lifetime of a fluorophore can be influenced by temperature, pH, oxygen concentration, and other environmental conditions. Within plant cells, the anthocyanin fluorescence lifetime correlates with distinct subcellular compartments. Vacuolar anthocyanins exhibit shorter fluorescence lifetime than the cytoplasmic pool. Consistent with these observations, lower pH of anthocyanins solutions correlated with shorter fluorescence lifetimes. We discuss here the use of FLIM as a tool for analyzing the subcellular distribution of anthocyanins and estimating variation in vacuolar pH in intact cells.