945 
SENSING OF PLANTS USING THE LASER-INDUCED FLUORESCENCE 
IMAGING SYSTEM 
M. Lang 1 , H. K. Lichtenthaler 1 , M. Sowinska 2 , P. Summ 2 , F. Heisel 2 , J. A. Miehe 2 and F. Tomasini 3 
! Botanisches Institut der Universität Karlsruhe, Kaiserstr. 12, D-76128 Karlsruhe, Germany 
2 Groupe d'Optique Appliquée, Centre de Recherches Nucléaires, 23 rue du Loess, F-67037 Strasbourg, France 
3 A.R.P., Centre de Transfert de Technologie, Route de Hausbergen, F-67088 Strasbourg, France 
ABSTRACT 
The blue-green and the red/far-red chlorophyll fluorescence emission of tobacco leaves (Nicotiana tabacum L.Jof a 
green form and an aurea mutant were examined applying a conventional spectrofluorometer and a new fluorescence 
imaging system consisting of a Nd:YAG laser (355 nm excitation), a high resolving CCD-camera, appropriate 
interference filters and processing equipment.The spectrofluorometer permitted to record the whole fluorescence 
emission spectra of selected leaf sections, which showed maxima or shoulders in the blue (F440), green (F520), red 
(F690) and far-red (F735) spectral regions and differences in the relative fluorescence intensities between leaf-veins 
and green vein-free leaf-regions. In contrast, the fluorescence imaging system was able to simultaneously sense the 
fluorescence bands of the whole leaves at distinct wavelengths (F440, F520, F690 and F735) and allowed a much 
finer differentiation between median and lateral leaf-veins and the intercostal fields. Fluorescence images of tobacco 
leaves precisely revealed the inverse contrast for the blue fluorescence and the chlorophyll fluorescence. The leaf- 
veins exhibited predominantly the blue-green fluorescence, whereas the vein-free intercostal regions showed 
primarily the red and far-red chlorophyll fluorescences. Consequently, the fluorescence ratios blue/red (F440/F690) 
and blue/far-red (F440/F735) were significantly higher in the main leaf-veins than in the vein-free intercostal field 
regions. The fluorescence ratios blue/red could easily be sensed with the fluorescence imaging system, giving precise 
images of the leaf which reflect the gradient in chlorophyll and carotenoid content within the leaf. The results indicate 
that the fluorescence imaging spectroscopy can be developed to a powerful method in the remoting sensing and stress 
detection of terrestrial vegetation. 
KEYWORDS 
Laser-induced Fluorescence, Fluorescence Images of Plants, Blue Fluorescence, Chlorophyll Fluorescence, 
Nicotiana tabacum L. 
INTRODUCTION 
The laser-induced fluorescence of terrestrial vegetation (LIF signatures) has been established as a tool in remote 
sensing in the past years in several laboratories. Excitation of plant leaves with UV-A radiation results in a 
fluorescence emission in the visible range of the spectrum with maxima in the blue region near 440 ± 10 nm (F440), 
green region near 520 to 530 nm (F520) and in the red and far-red region near 684 to 690 and 735 to 740 nm (F690 
and F735) (Chappelle et al., 1984; Lang and Lichtenthaler, 1991; Lang et al., 1992; Lichtenthaler et al., 1992; Stober 
and Lichtenthaler, 1992). The red and far-red chlorophyll fluorescence emission and its variation in leaves had been 
studied for many years (Virgin, 1954; Kochubey et al., 1986; Lichtenthaler, 1987a; Lichtenthaler and Buschmann, 
1987; Lichtenthaler and Rinderle, 1988). The blue-green fluorescence emission of leaves, in turn, appears to be a 
complex signal from various plant phenolics like hydroxycinnamic acids, coumarins, catechin and their derivatives 
(Goulas et al., 1990, Lang et al., 1991), which can change from plant to plant. After peeling off the epidermis of 
tobacco leaves the intensity of the blue-green fluorescence was reduced, demonstrating the epidermis as a major 
source of the blue-green fluorescence of leaves (Lang et al., 1991). Studies with the fluorescence microscope 
confirmed these findings, but showed in addition that cell walls are the origin of the blue-green fluorescence and that 
besides the epidermis cells also the leaf-veins exhibit a high blue-green fluorescence emission (Stober and 
Lichtenthaler, 1993a). From studies with white and green leaves it was concluded that the blue-green fluorescence 
emitted by the green mesophyll cells is reabsorbed by the chlorophylls and carotenoids in the chloroplast-containing