947
Fluorescence imaging spectroscopy: Fluorescence images were sensed with a digital camera developed by A.R.P.,
Strasbourg, in collaboration with the Centre d'Etudes Nucléaires, Strasbourg.The fluorescence was excited with a
mode-locked, Q-switched cavity dumped tripled Nd:YAG laser (A. exc = 355 nm, FWHM» 100 ps, frequency of
repetition 1 kHz, energy« 10 uJ/pulse. A diffused excitation beam was applied in order to illuminate the whole leaf
surface. In order to simulate the outdoor situation, the measurements were performed of illuminated leaves, which
were in the steady state of the chlorophyll fluorescence induction kinetics by application of additional white light
before and during the measurements. The fluorescence images were sensed at a distance of 50 cm and focussed via a
lens on the image intensifier, that was gated (gate width 20 ms) to reduce the influence of the ambient light (ARPAnimater
(ARPAnimater V2, ARP, Strasbourg, France). Four different bandpass filters (10 nm bandwidth) were used for the
recording of the fluorescence images at 440, 520, 690 and 735 nm, which approximately correspond to the
fluorescence maxima/shoulders in the blue (F440), green (F520), red (F690) and far-red (F740) spectral regions. The
fluorescence images were corrected for the equipment and processed with the ARP-Software "Animater". With the
described equipment the fluorescence images could also be sensed from a distance of several meters. The imaging
system applied here can easily be further developed for the remote sensing of plants by using more sensitive
detectors.
RESULTS
The leaves of the green tobacco su/su contained a higher pigment content (chlorophylls a+b: 45 pg cm -2 ; carotenoids
x+c: 8.7 pg cnr 2 leaf area) and different pigment ratios (chlorophylls a/b = 2.7 and chlorophylls/carotenoids =
(a+b)/(x+c) = 5.2) than the yellowish-green leaves of the aurea tobacco Su/su (chlorophylls a+b: 19.5 pg cm- 2 ;
carotenoids x+c: 5.4 pg cm- 2 leaf area; pigment ratios: a/b = 3.3 and (a+b)/(x+c) = 3.6). The UV-A induced
fluorescence emission spectra obtained with a conventional fluorometer of the leaves of green tobacco and the
chlorophyll-poor aurea mutant Su/su showed considerable differences when measured in leaf-vein regions or in the
vein-free intercostal fields as shown for the lower leaf-side in Fig.l. For green and aurea tobacco the blue
fluorescence (F440) was significantly higher (p < 0.05) in the region of the main leaf-veins than the F440 in the
intercostal fields. In the case of the vein-free intercostal fields, the red and the far-red chlorophyll fluorescence
intensities F690 and F735 were higher in both tobacco forms than the blue-green fluorescences (F440 and F520) .
Due to its higher chlorophyll content, the green tobacco exhibited a higher chlorophyll fluorescence yield in the
intercostal field than the aurea mutant. In the chlorophyll-poor aurea-mutant the blue-green fluorescence of the leafvein
leafvein was higher than the red chlorophyll fluorescence, which however, did not apply to the green tobacco leaf with its
higher chlorophyll and carotenoid content. These differences in fluorescence yield for the four spectral regions could
be quantified by forming the different fluorescence ratios blue/red, blue/far-red, red/far-red and blue/green of upper
and lower leaf-sides of green and aurea tobacco leaves as listed in Table 1. The fluorescence ratio blue/red
(F440/F690) was significantly higher for the main leaf-veins than for the intercostal fields of both green and aurea
tobacco.
Also the two chlorophyll fluorescence emissions, F690 and F735, showed differences between leaf-vein and
intercostal fields of aurea and green tobacco, which are due to a different chlorophyll content per leaf area unit and a
partial reabsorption of the 690 nm fluorescence emission by the in vivo chlorophyll. In the main leaf-veins of both
aurea and green tobacco the red fluorescence F690 exhibited a clear maximum, whereas the much lower far-red
fluorescence band F735 showed up only as a shoulder. Intercostal fields, in contrast, exhibited a clear maximum at
735 nm, and the intensity of F690 was either significantly lower than the F735 band (green tobacco) or higher (aurea
tobacco) (Fig. 1). These differences in the expression of the two chlorophyll fluorescence bands could be quantified
in the fluorescence ratio F690/F735 (Table 1).
In a further experiment we studied the distribution of the blue-green and red/far-red fluorescence signatures of
tobacco leaves by recording the fluorescence emission of all leaf sections along the transverse axis of the lower leafside
leafside of an aurea tobacco leaf. The results are visualized in a 3-d surface plot of 7 fluorescence emission spectra from
all transverse leaf regions (Fig. 2A). Each fluorescence spectrum was calculated as the average of 4 independent
spectra measured from two different tobacco plants. Leaf region No. 4, the central part of the leaf with the main leafvein,
leafvein, did not only show the strongest blue-green fluorescence emission, but also the highest chlorophyll fluorescence
emission in the 690 nm region. Leaf regions No. 2 and No. 5 represent areas of the intercostal fields with lateral leafveins,
leafveins, where the blue-green fluorescence was significantly higher than in the vein-free intercostal fields (leaf regions
3, 6 and 7), but not as high as in the main leaf-vein No. 4. The blue-green fluorescence emission was found to be
higher in the regions of the main leaf-vein and the side leaf-veins, reflecting their lower chlorophyll and carotenoid
content per leaf area unit, than in the intercostal fields. A disadvantage of the conventional fluorometers is the fact
that the transition between intercostal fields and main leaf-vein cannot be resolved, since the fluorescence signal can
only be measured of a larger leaf section. The method of scanning a leaf by successive measuring individual leaf
