In vivo label-free mapping of the effect of a photosystem II inhibiting herbicide in plants using chlorophyll fluorescence lifetime

Background: In order to better understand and improve the mode of action of agrochemicals, it is useful to be able to visualize their uptake and distribution in vivo, non-invasively and, ideally, in the field. Here we explore the potential of plant autofluorescence (specifically chlorophyll fluorescence) to provide a readout of herbicide action across the scales utilising multiphoton-excited fluorescence lifetime imaging, wide-field single-photon excited fluorescence lifetime imaging and single point fluorescence lifetime measurements via a fibre-optic probe. Results: Our studies indicate that changes in chlorophyll fluorescence lifetime can be utilised as an indirect readout of a photosystem II inhibiting herbicide activity in living plant leaves at three different scales: cellular (similar to mu m), single point (similar to 1 mm(2)) and macroscopic (similar to 8 x 6 mm(2) of a leaf). Multiphoton excited fluorescence lifetime imaging of Triticum aestivum leaves indicated that there is an increase in the spatially averaged chlorophyll fluorescence lifetime of leaves treated with Flagon EC-a photosystem II inhibiting herbicide. The untreated leaf exhibited an average lifetime of 560 +/- 30 ps while the leaf imaged 2 h post treatment exhibited an increased lifetime of 2000 +/- 440 ps in different fields of view. The results from in vivo wide-field single-photon excited fluorescence lifetime imaging excited at 440 nm indicated an increase in chlorophyll fluorescence lifetime from 521 ps in an untreated leaf to 1000 ps, just 3 min after treating the same leaf with Flagon EC, and to 2150 ps after 27 min. In vivo single point fluorescence lifetime measurements demonstrated a similar increase in chlorophyll fluorescence lifetime. Untreated leaf presented a fluorescence lifetime of 435 ps in the 440 nm excited chlorophyll channel, CH4 (620-710 nm). In the first 5 min after treatment, mean fluorescence lifetime is observed to have increased to 1 ns and then to 1.3 ns after 60 min. For all these in vivo plant autofluorescence lifetime measurements, the plants were not dark-adapted. Conclusions: We demonstrate that the local impact of a photosystem II herbicide on living plant leaves can be conveniently mapped in space and time via changes in autofluorescence lifetime, which we attribute to changes in chlorophyll fluorescence. Using portable fibre-optic probe instrumentation originally designed for label-free biomedical applications, this capability could be deployed outside the laboratory for monitoring the distribution of herbicides in growing plants.