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. 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 two 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.
