Fluorescence lifetime of plant leaves with sub-nanosecond resolution

Abstract

The study aimed to develop a measurement apparatus for in vivo chlorophyll-a (Chl-a) fluorescence decay measurements of plants by means of Time Correlated Single Photon Counting (TCSPC). In this approach, sub-nanosecond laser pulses with a repetition rate of 10 MHz are applied to excite the sample, followed by the analysis of arrival times of the emitted fluorescence photons. Photon statistics are generated by iteratively fitting the sum of 2 exponential functions. The tool was tested on both plastid and in vivo leaf samples of Savoy cabbage (Brassica oleracea var. sabauda) with 3-4 subsequent leaves giving a complete sample coverage starting from the outermost. The Chl-a fluorescence lifetime exhibited a gradual increase in both the isolated plastid suspensions and the in vivo leaf samples towards the innermost leaf layers explained by an increase of natural absence of light (etiolation syndrome). Furthermore, cadmium stress and iron deficiency were investigated on treated sugar beet (Beta vulgaris) samples in vivo using TCSPS measurements. The reduced fluorescence quenching resulted in an increased fluorescence lifetime. Finally, a long-term (10 week) testing of the setup was carried out on Chl-retaining resurrection Haberlea rhodopensis plants protecting themselves by an elevated non-photochemical quenching yielding a decrease of fluorescence lifetime during their desiccation.