Application of fluorescence lifetimes to multi-imaging analysis in plant cells

Multi-imaging analysis has become an indispensable technique to visualize multiple target proteins and intracellular components simultaneously. While current multi-imaging analysis relies on the differences in emission spectra of fluorescent molecules, the use of fluorescence lifetime imaging microscopy (FLIM), which exploits the differences in fluorescence lifetimes of fluorescent proteins, in multi-imaging analysis is quite limited. In this study, we successfully discriminated fluorescent proteins with similar colors but different fluorescence lifetimes in vitro and in planta. We found that four fluorescent proteins with similar emission spectra could be distinguished by FLIM. In addition, we found that FLIM could clearly separate fluorescent proteins if they differ by at least 0.2 ns. In a proof-of-concept experiments for plant live imaging, we transiently expressed fluorescent proteins with different subcellular localization tags in Physcomitrium patens by particle bombardment. Each fluorescent protein exhibited its fluorescence lifetime at the subcellular localization corresponding to the localization tag in P. patens with little or no effect of chlorophyll autofluorescence. Our results demonstrate the effectiveness of FLIM in revealing the spatiotemporal dynamics of a large number of fluorescent proteins in living plant cells.