3-RESULTS 
3.1.Decay-associated spectra 
For comparative purposes we first measured the excitation and emission DAS for standard free 
nucleotides (in water) in the same apparatus and temperature (20°C) as for leaves. The results of this analysis is 
presented in Table 1. As expected for single fluorophores, the individual lifetimes were homogeneous, 
wavelength independent, throughout the excitation and emission spectrum, therefore only results of global 
analysis are presented. The calculated lifetimes agrees with the reported values in the literature [19, 20]. The 
results on the lifetime and the number of resolved components for standards validated the convolution algorithm 
and permitted to classify these potential leaf fluorophores by lifetime classes. 
Emission DAS of leaves were recorded with the fixed excitation wavelength at 360 nm. First, an 
individual analysis was performed for each decay .The same distribution of fractional intensities was obtained 
either by individual or global analysis of decays of a single scan. Results of the global type of analysis are 
presented in Fig. 1 B for the abaxial side of a sugar beet leaf. In Fig. 1 C, spectra from two samples of 
mesophyll (abaxial side) were combined, and in Fig. 1 A, spectra from four samples of leaves (adaxial side) were 
combined. Although the scatter of points was increased by combining spectra of different samples it demonstrates 
the reproducibility of the method and avoided the effects of sample aging (excessive UV-illumination). 
From the presented spectra it can be seen that in intact leaves the fast and medium components are 
dominated by blue emitting fluorophores (maximum at 440-450 nm). As these components contribute up to 
40 % each to the total fluorescence in the blue region (Table 2) the average fluorescence lifetime is strongly 
decreased in the blue region of the spectrum compared to the green, in intact leaves (Fig. 1 A and B). This effect 
is not seen in leaves without epidermis, where the mesophyll is directly exposed to illumination (Fig. 1 C). In 
leaves, the fast and medium components have very similar spectra and thanks to their large contributions, they 
define the form of the total fluorescence spectrum. Still, in the two slower components the contribution of the 
green emitting fluorophores becomes far more important. The fluorescence intensity of the very slow component 
at the green maximum was even higher than at the blue. In addition, there was a shift of the blue maximum to 
longer wavelengths, towards 460 nm. 
In the mesophyll (Fig. 1 C), the blue maximum was at 460 nm for all four components and the average 
lifetime of fluorescence was wavelength independent. This indicates that the the major blue emitting fluorophores 
with short-lived fluorescence, were eliminated with the epidermis. Moreover, it could suggest that the slow and 
very slow component in intact leaves could emanate from the same or similar fluorophores as in the mesophyll. 
In Fig. 1 C, it can also be seen that the slow component in the mesophyll is dominated by a green (525 nm) 
emitting fluorophore and the very slow component dominated by a blue (460 nm) emitting fluorophore, with 
maxima corresponding to flavin nucleotide (525 nm) and nicotinamide nucleotide (460 nm), respectively. 
Table 1. Fluorescence lifetime and spectral characteristics of nicotinamide and flavin nucleotides, and 
riboflavin. 
NADPH 
FAD 
FMN 
riboflavin 
excitation 
260 
270 
270 
270 
maxima (nm) 
340 
375 
375 
375 
450 
450 
450 
emission 
maximum (nm) 
460 
525 
525 
525 
average 
lifetime (ns) 
0.39 
2.87 
4.56 
4.61 
kinetic 
component 
t (ns)* /(%) 
t(ns) /(%) 
t(ns) /(%) 
t(ns) 
/(%) 
very fast 
0.10 26 
0.02 
2 
— — 
— 
— 
fast 
0.45 72 
— 
— 
0.90 2 
0.38 
2 
medium 
2.29 2 
2.42 
71 
— — 
— 
— 
slow 
— — 
4.30 
27 
4.62 98 
4.61 
98 
* Fractional intensities were defined as: f[ = CXj Ti / Z Cti X \, 
and the average lifetime as: x m = ^ /j Xj = £ Oti Ti 2 / £ CXJ Ti , 
where Otj represent the pre-exponential factors of the decay function.