946 
mesophyll cells due to the spectral overlapping of the absorption bands of the pigments with the blue-green 
fluorescence of the cell walls. The decrease of the blue-green fluorescence during greening of etiolated wheat leaves 
with increasing content of chlorophylls and carotenoids confirmed the partial reabsorption of emitted fluorescence 
is still in discussion (Chappelle et al, 1991; Cerovic et al., 1993), but it was clearly demonstrated that intact isolated 
chloroplasts did not exhibit a blue-green fluorescence signal (Lang et al., 1992) which excludes chloroplasts as a 
source of the blue-green fluorescences. Furthermore, the red chlorophyll fluorescence exhibited the Kautsky effect 
(Kautsky and Hirsch, 1934; for original references confer the review of Lichtenthaler, 1992), whereas the blue-green 
fluorescence was constant during the fluorescence induction kinetics (Stober and Lichtenthaler, 1993b). 
The changes in relative fluorescence intensities of blue-green fluorescence bands and the red/far-red chlorophyll 
fluorescence bands can be expressed by several fluorescence ratios, such as blue/red (F440/F690), blue/far-red 
(F690/F740), red/far-red (F690/F735) and blue-green (F440/F520). The ratios seem to be typical for a particular stage 
in leaf development, pigment content as well as growth and environmental conditions and thus provide essential 
information on the physiological state of plants. Therefore it is of interest to know these fluorescence ratios and to 
apply them in the remote sensing of plants. 
Between the chlorophyll fluorescence ratio F690/F735 and the in vivo chlorophyll content of leaves there exists an 
inverse relationship (Lichtenthaler and Buschmann, 1987; Lichtenthaler and Rinderle, 1988). Since all long-term 
stress events reduce the chlorophyll content of leaves, fhe fluorescence ratio F690/F735 can be applied as an indicator 
of stress of terrestrial vegetation (Lichtenthaler and Rinderle, 1988; Hâk et al., 1990). The ratio of blue/green 
fluorescence, (F440/F520), linearily correlates to the pigment ratio of chlorophylls to carotenoids (a+b)/(x+c) and 
thus provides complementary information on the carotenoid content of leaves (Stober and Lichtenthaler, 1992). The 
fluorescence ratio blue/red (F440/F690) increased with increasing age of spruce needles and under water stress 
(Lichtenthaler et al., 1991) as well as with increasing irradiance during the growth of plants (Stober and 
Lichtenthaler, 1993c). 
Here we report on the comparison between fluorescence emission spectra of leaf sections measured in a conventional 
spectrofluorometer and the fluorescence images obtained by fluorescence imaging spectroscopy of particular 
fluorescence bands. Although spectrofluorometers can be applied to determine the fluorescence emission spectra of 
the leaf sections, it is not possible to record simultaneously the fluorescence emissions of a complete leaf or obtain 
information on the fluorescence intensity of a small point on the leaf surface. In contrast, digital cameras permit the 
recording of fluorescence images of whole leaves or plants and also contain fluorescence information of very small 
leaf spots. Fluorescence images thus provide essential information on the gradient of the blue-green and the red 
chlorophyll fluorescence within the leaf, a knowledge which is needed before this technique can be applied for the 
remote sensing of plants. 
Plants and pigment determination: Tobacco plants (Nicotiana tabacum L., green form su/su and aurea mutant 
Su/su; Schmid 1971) were grown in the greenhouse of the Botanical Garden of the University of Karlsruhe for 4 
months at an ambient temperature of 22° C ± 4 on a peat containing mineral nutrient (TKS II) . The length of the 
Lichtenthaler (1987) applying the spectrophotometer UV2001-PC (Shimadzu, Duisburg, Germany). 
Spectrofluorometry: Fluorescence emission spectra of tobacco leaves were performed on the LS-50 
Spectrofluorometer (Perkin-Elmer, Überlingen, Germany). For the comparison we used the same excitation 
wavelength (355 nm) as used in the fluorescence imaging system, since different excitation wavelengths applied for 
normalizing at the excitation wavelength (355 nm) and for the instrument equipment using the supplier's correction 
factor spectrum. The image of the measured leaf strip was created by scanning the leaf sections into a micro 
computer ( Scan Jet lie, Hewlett Packard, Germany) and processing with the software package Corel Draw (Corel 
Corp., Canada). 
(Stober and Lichtenthaler, 1992). A small contribution of chloroplast-bound NADPH to the blue fluorescence signal 
MATERIAL AND METHODS 
tobacco leaves was 15 - 20 cm (green form) and 13-17 cm (aurea mutant). The determination of the photosynthetic 
pigments (chlorophylls (a+b) and total carotenoids (x+c)) of the tobacco leaves was performed by the method of 
fluorescence emission spectra result in a different shape of the fluorescence spectra. Spectral bandwidth was 2.5 nm 
for the excitation and 10 nm for the selective excitation of leaf-veins. The fluorescence was excited at an angle of 60 
and sensed at an angle of 30° to the leaf surface . A 340 nm bandpass filter was placed into the excitation beam and a 
430 nm cut-off filter into the fluorescence emission beam. The fluorescence spectra were corrected for reflections by