949 
Table 1 Differences in the fluorescence ratios blue/green (F440/F520), blue/red (F440/F690), blue/far-red 
(F440/F735) and red/far-red (F690/F735) between main leaf-veins and vein-free intercostal fields. The original 
spectra are shown in Fig. 1 and Fig. 2. Mean of 4 determinations from 2 leaves, standard deviation 5 % or less. 
F440/F690 
F440/F735 
F690/F735 
F440/F520 
Green tobacco su/su 
Upper leaf-side 
main leaf-vein 
0.21 
0.20 
0.93 
1.08 
intercostal field 
0.07* 
0.05* 
0.56* 
1.75* 
Lower leaf-side 
main leaf-vein 
0.67 
1.19 
1.79 
1.92 
intercostal field 
0.03* 
0.03* 
0.88* 
1.11* 
Aurea tobacco Su/su 
Upper leaf-side 
main leaf-vein 
1.23 
2.27 
1.80 
1.58 
intercostal field 
0.66* 
0.62* 
0.78* 
1.65 
Lower leaf-side 
main leaf-vein 
1.50 
2.55 
1.73 
1.45 
intercostal field 
0.70* 
0.90* 
1.29* 
2.05* 
* These fluorescence ratios of intercostal fields are significantly different from those of the main leaf vein (p <0.01) 
sections in a conventional spectrofluorometer is not fully accurate, because it represents the sum of fluorescence 
signals of the measured leaf section. 
Imaging spectroscopy, in contrast, is more precise, since it permits the simultaneous fluorescence screening of the 
whole leaf area for a particular fluorescence band in the blue, green or red spectral region. Fluorescence images for 
F440 and F690 of the lower leaf-side of a young green tobacco leaf are shown in Fig. 3, where the bright parts 
indicate a high fluorescence yield. The fluorescence images clearly demonstrated that the blue fluorescence F440, 
shown in Fig. 3A, emanated primarily from the main and lateral leaf-veins, whereas the vein-free intercostal fields 
were found to be the major source of the red chlorophyll fluorescence F690 (Fig. 3B). Fig. 3 also shows that blue 
fluorescence and the red fluorescence give inverse contrast images of the tobacco leaf. This also applies to the green 
fluorescence F520, which comes also mainly from the leaf-veins and negatively contrasts the far-red chlorophyll 
fluorescence F735. 
From the fluorescence images sensed in the blue, green, red and far-red spectral regions (F440, F520, F690 and F735) 
one can, with the help of the processing software, create an image of the tobacco leaf which is based on the various 
fluorescence ratios. This is shown for the fluorescence ratio blue/red (F440/F690) in Fig. 4. In this image bright 
points and lines signalize higher values for the fluorescence ratio blue/red. It is evident that the blue/red fluorescence 
ratio is highest in the main leaf-vein and decreases via lateral leaf-veins to much lower values in the vein-free 
intercostal fields. 
DISCUSSION 
Excitation with UV-A radiation (355 run) either from the lamp of a conventional spectrofluorometer or by a Nd:YAG 
laser resulted in a blue-green and red + far-red fluorescence emission of plant leaves. The full fluorescence spectra of 
leaves of green and aurea tobacco showed maxima or shoulders in the blue (F440), green (F520), red (F690) and far- 
red spectral region (F735) as had been shown before for green leaves of other plants (Chappelle, 1984; Lang and 
Lichtenthaler, 1992; Lichtenthaler et al., 1992). That the blue-green fluorescence and the red chlorophyll emission are 
not evenly distributed over the whole leaf surface but show an inverse gradient, has been demonstrated here for the 
first time. These differences can easily be quantified by forming the different fluorescence ratios blue/red, blue/far- 
red, red/far-red and blue/green, which are quite different for the individual parts of the leaf surface.