Remote Sensing Vegetation Status by Laser-Induced Fluorescence

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Remote Sensing Vegetation Status by

Laser-Induced Fluorescence

K. P. Gunther, H.-G. Dahn, and W. Liideker

°°

I n November 1989 the EUREKA project LASFLEUR (EU 380) started as an European research effort to investi- gate the future application of far-field laser-induced plant fluorescence for synoptic, airborne environmental moni- toring of vegetation. This report includes a brief introduc- tion in a theoretically approach for the laser-induced fluorescence signals of leaves and their spectral and radio- metric behaviour. In addition, a detailed description of the design and realization of the second generation of the far-field fluorescence lidar (DLidaR-2) is given with special regard to the optical and electronical setup, fol- lowed by a short explanation of the data processing.

The main objectives of the far-field measurements are to demonstrate the link between laser-induced fluorescence data and plant physiology and to show the reliability of remote single shot lidar measurements. The data sets include the typical daily cycles of the fluorescence for different global irradiation. As expected from biophysical models, the renugtely sensed chlorophyll fluorescence is highly correlated with the carbon fixation rate, while the fluorescence ratio F685 /F730 is only dependent on the chlorophyll concentration. Drought stress measurement of evergreen oaks Quercus pubescens confirm the find- ings of healthy plants with regard to the fluorescence ratio F685/F730 while the fluorescence signals of stressed plants show a different behavior than nonstressed plants.

Additionally, the corresponding physiological data (poro- meter and PAM data) are presented.

INTRODUCTION

From an optical point of view healthy vegetation is characterized by the highly variable green color of the

* Institute of Optoelectronies, DLR-Oberpfaffenhofen, Wessling, Germany

Address correspondence to K. P. Giinther, Institute of Optoelec- tronics, DLR-Oberpfaffenhofen, M/inchenerstr. 20, D-82230 Wess- ling, Germany.

Received 18 October 1992; revised 1 May 1993.

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leaves and needles. This optical impression of vegetation is mainly correlated with the absorption features of the different plant pigments (e.g., chlorophyll a and b) (Gates et al., 1965), while the intensity of the green color is closely linked to the internal pigment concentration of the leaf or needle and the external structuring of the canopy, often referred as leaf area index (LAI) and leaf angle distribution (Curran et al., 1983).

During autumnal breakdown (see, e.g., Boyer et al., 1988), or due to severe environmental stress, the green color of vegetation changes to yellow or brown because the internal concentration of the pigments will be reduced. In addition, the external structure of the vegetation becomes more sparse compared to the very dense package of leaves and needles on healthy branches of trees. Thus the influence of the soil augments and changes the overall color of vegetation for a remote observer.

These known effects are used since some decades for vegetation monitoring from aircrafts or satellites by operating multispectral scanner, imaging spectrometer, (Vane and Goetz, 1988), or false color infrared photogra- phy. The benefits of these techniques are broad. A lot of qualitative knowledge about regional and global ecosystems as well as about ecosystem changes is based on those observations. On the other hand, the quantitative evaluation of multispectral reflectance data, for example, in terms of different classes of damage, always needs an intensive ground truth observation by experi- enced investigators in order to train the classification algorithms (Kritikos et al., 1988). Though the possibility of the determination of useful parameters for the estimation of concentrations of chlorophyll a, chlorophyll b, and carotinoides could be derived in the lab by Chappelle et al. (1992). It was not yet possible to derive a quantitative map of a region showing, for example, the variability of physiological parameters of vegetation from remote reflectance data.

Beside the color, the state of vegetation can be quantified by internal physiological parameters such as

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pigment concentration, photosynthetic activity, stomatal resistance, or leaf water content. From a more application-oriented point of view, a quantification of plant status in terms of nutrient uptake, growth rate, or water deficiency is needed at an early stage long before some stress has expressed a visible symptom such as discolor- ation.

One technique to observe the physiological status of leaves or needles by optical methods is based on the fluorescence of the plant pigments, mainly the chloro- phylls [see as a review, e.g., Lichtenthaler (1988)]. But up to now, most investigations on chlorophyll fluorescence were done in the laboratory with suspensions of chloroplasts to understand the internal biomolecular processes of photosynthesis. In the laboratory, the well- defined preparation of samples (e.g., predarkening) fa- vored complex and time-consuming measurements such as the pulse-amplitude-modulated technique (PAM) introduced by Schreiber et al. (1986). The PAM technique is an extension of the conventional one- or two-wavelength Kautsky induction measurement (e.g., Franck et al., 1969;

Lichtenthaler and Rinderle, 1988).

For synoptic, remote measurements of the physiological status of plants under environmental conditions, new techniques are needed such as those introduced by Hoge et al. (1983), Chappelle et al. (1984a,b; 1985), and Zimmermann and Gfinther (1986). The first ap- proaches are oriented towards the laser-induced fluorescence spectroscopy to derive from spectral information parameters related to the physiology of plants (Zimmermann and Gfinther, 1986; Lichtenthaler et al., 1986).

PLANT FLUORESCENCE AND PHYSIOLOGY In the following, only a short presentation will be given demonstrating the relation of leaf fluorescence and leaf physiology. The leaf fluorescence F(~,) (see Fig. 1) can be written as (Dahn et al., 1992)

F(~,) = I0(~-L) x c x a*(,~L) × • x Ie -(k+~)z dz, with

Io(2L) = laser intensity,

c = concentration of chlorophyll in the leaf, a*(;tL) = specific absorption coefficient of chlorophyll at

laser wavelength,

= fluorescence efficiency at detection wavelength,

k,k'-- extinction coefficients at laser and detection wavelength.

The exponential function is integrated over the leaf thickness d. For the derivation of F(2), a homogeneous leaf of thickness d containing only chlorophyll pigments was assumed; but, without discussing the implications of this

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Figure 1. Typical fluorescence spectrum of a maize plant, excited with a tripled Nd:YAG laser at 355 nm and detected with an optical multichannel analyzer (OSMA from SI). The plants were grown in a phytochamber.

restriction, the principal features of the leaf fluorescence F(;t) can be shown to establish the relationship between fluorescence and physiology.

As can be seen, the fluorescence of chlorophyll depends on parameters which can be measured such as laser intensity and the specific absorption coefficient and on parameters which are ab initio unknown such as chlorophyll concentration and fluorescence efficiency.

For an understanding of the variability of plant fluorescence and the correlation to plant physiology, all parameters related to F(2) should be determined. In principle, this approach is possible in the laboratory or in field experiments from the ground, but problems arise for remote measurements from aircrafts. Therefore, one has to look for general findings or reasonable assump- tions with regard to optical constants of leaves, fluorescence efficiency, and chlorophyll concentration.

From a theoretical point of view, Dahn et al. (1992) could show that even from a remote platform chlorophyll concentration can be determined by monitoring the chlorophyll fluorescence band at two different wave- lengths. This finding was confirmed by several experiments [see Hak et al., 1990]. Thus one can conclude that leaf fluorescence F(~.) still depends in an unknown behavior on fluorescence efficiency and optical leaf parameters (extinction coefficients and leaf thickness).

If the fluorescence measurements are restricted to investigations on leaves with chlorophyll concentrations above = 10/~g/cm 2, one can show that the extinction coefficient at the fluorescence maximum is mainly determined by the specific absorption coefficient and is insen- sitive to the scattering coefficient. In this regime, the integral of formula (1) becomes nearly independent of the leaf thickness and can be represented by the inverse of the chlorophyll concentration and the specific absorp-

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tion coefficient. Thus, the measured fluorescence intensity will be determined by fluorescence efficiency, the ratio of the specific absorption coefficients at excitation and emission wavelength, and, of course, by the laser intensity.

At the end of this short introduction to a quantitative description of laser-induced leaf fluorescence, the ques- tion still remains: How is the fluorescence of chlorophyll related to the physiology of plants?

Most of the light absorbed by the pigments of plants (chlorophyll a and b, carotinoids) will be used for photosynthesis in order to maintain and optimize the growth under different environmental conditions. Only a small portion of the absorbed light, typically less than 3%, is emitted as chlorophyll fluorescence or heat, but, due to this very close link between chlorophyll fluorescence and photosynthesis, it is possible to monitor photosynthesis optically. For an intact photo system an inverse relation between the emitted chlorophyll fluorescence and the rate of photosynthesis exists (Renger, 1982).

T E C H N I C A L C O N C E P T O F DLidaR-2

At the beginning of the LASFLEUR project, a laboratory fluorescence lidar was set up for monitoring the fluorescence spectra of leaves in a high resolution mode from 400 nm to 800 nm in single shot operation. The system was successfully operated in the near-field (up to a distance of 5 m between the system and the target) with a variable laser divergence of up to 10 mrad. Thus it was possible to investigate not only single leaves but also ensembles of leaves for averaging the fluorescence of leaves with different orientations, distances, and physiological conditions (G/inther et al., 1991; Dahn et al., 1992).

Pushed by the requirement that a far-field fluorescence lidar should also operate with a laser divergence capable of illuminating the crown of a tree from a remote platform (aircraft), it was concluded that for the development of the far-field lidar a concept different from the optical multichannel analyzer in connection with a monochromator must be used.

From our measurements as well as from other data (Chappelle et al., 1984a,b; Lichtenthaler and Rinderle, 1988), it was concluded that a fluorescence lidar system with four spectral detection channels is sufficient to monitor the red chlorophyll fluorescence and the blue fluorescence from other leaf pigments.

For the excitation of the leaf fluorescence a tripled, air-cooled Nd-YAG laser (Spectra Physics, DCR11) is used. The UV light of the laser is suitable to excite the blue fluorescence and the red fluorescence of chlorophyll as seen in Figure 1. The fluorescence lidar is configured as a coaxial system with a variable beam expander just in front of a Cassegrain telescope (CE- LESTRON C8 with a mirror 20 cm in diameter). Due

to the movable collecting mirror of the telescope, the focal length of the optic can be varied from 75 cm to 203 cm in order to adjust the image size. Instead of complex wavelength separation optics based on dichroic mirrors and lenses for a parallel beam, a fiber optic was coupled directly to the output of the telescope. The entrance aperture of the fiber bundle is 18 mm in diameter with exact fitting to the exit aperture of the telescope while the exit of the fiber bundle is spliced up into four smaller fiber bundles. Due to this unique design, the image information at the entrance of the fiber optic is lost whereby the intensity is split according to the ratio of the diameter of the exit bundle to the entrance bundle.

For adjusting the exit aperture of the fiber optics to the entrance diameter of the photomultiplier, a mirrored cone was constructed and attached to the photomultiplier housings. These housings also contain the small band interference filter, the short pass filter and the edge filter for wavelength separation. During the operation of the system, it was demonstrated that this optical design results in a rugged and easy operation without complex adjustments.

As mentioned, blue and red enhanced photomultiplier were used as detectors. To increase the dynamic range of the lidar measurements under daylight conditions, the charge flow through the dynode cascade has to be low, due to saturation effects of the PMT during continuous measurements. This is realized by gating the mulitplier for the short period of the fluorescence signal which is determined by the laser pulse duration and the maximum height of the vegetation (typically, 50 ns to 200 ns). By switching the voltage at the first dynode between ground and the voltage level of the divider chain, it is possible to reduce very efficiently the charges produced by intense solar background. Based on the multiplier gain equation, a simple approach is given to estimate the "HOLD-OFF" ratio (Barrick, 1986):

with

G = K*V ~n,

G = multiplier gain

K = constant (depends on cathode) V = multiplier voltage

a = efficiency of dynode amplification n = number of dynodes (dl-dn)

One can calculate the charge flow suppression of at least a factor of G(n = 8;dlon) / G(n = 7;dlo~) = 240 assuming a multiplier voltage of ~-- 1500 V and a typical a of 0.75.

The actual value for the H O L D - O F F ratio is expected to be quite higher, because the ground-switched first dynode dl changes the electron optical properties of the PMT. Additionally the dynode is not physically removed from the multiplier and acts as a primary electron absorber. Both effects are not regarded in the

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equation. From the literature (Barrick, 1986) HOLD- O F F ratios in the order of 103-104 are well known.

From our data the H O L D - O F F ratio is estimated to be

>104 , limited by the resolution of our system. If the multiplier is detecting a measurable signal with dl switched off, the signal is definitely nonlinear if the same number of photons is reached in the gate (dl switched on) under the same gain conditions.

If the cathode of the multiplier is set to ground while the anode is set to the maximum positive voltage, it is sufficient to switch about 200 V related to ground instead of 200 V biased by the high negative voltage usually connected to the cathode (typical 1.5 kV). For security reasons, the output of the photomultiplier must be connected to a HV capacitor in order to separate the high potential of the multiplier and the potential of the electronics. The gate electronic was designed and realized by DLR and Dressier HF.

For ground operation the photomultiplier signals are fed to a fast digitizing four-channel oscilloscope (Tektronix, TDS 540) with a digitizing rate of 250 Mega samples per second and channel and an analogue band- width of 500 MHz. The resolution is 8 bits. The digital data of the oscilloscope are transferred to the computer via an IEEE 488 interface. The data transfer is restricted to a lower repetition rate than the laser repetition rate of 12 Hz.

For static measurements from the ground it is possible to average the fluorescence signals in the oscilloscope before the data transfer to the storage device. In this mode a higher temporal resolution can be achieved up to sample rates of 1 Giga samples per second and channel. Operating the detection electronics with this high resolution, time-resolved measurements for analyz- ing structural phenomena of the plants can be achieved.

The correct timing and triggering of the lidar system is realized by different CAMAC gate and delay units (Le Croy). As additional information for data processing the laser pulse energy, the high voltage settings of each photomultiplier (gain) and the external global irradiance are digitized (12 bits) and stored for each laser shot.

The schematic setup of the fluorescence lidar DLidaR-2 is shown in Figure 2.

All results presented here were obtained during the international LASFLEUR campaign performed in October 1991 in Viterbo and organized by the Forest Department of the University of Tuscia. The main goal of this campaign was to operate different fluorescence lidars in order to

• investigate the daily cycle of plant fluorescence,

• correlate far-field lidar data with physiological measurements,

• correlate far-field lidar data with reflectance measurements.

BEAM EXPANDER

TELE

SCOPE 5-CHANNEL OPTICS PHOTO- MULTIPLIER I ENERGY-MONITOR

1386 MS-DOS i COMPUTER I LASER-TRIGGER

Figure 2. Schematic setup of the fluorescence lidar DLidaR-2.

DLidaR-2 DATA PROCESSING

In Figure 3, typical time-resolved fluorescence signals of the four spectral channels are shown. The time scale is chosen to illustrate the opening of the photomultiplier gate at time 0 and its closing about 160 ns later. The peaks at that times show the interference induced by switching the high voltage on and off. One can observe that the fluorescence signals of the red channels (685 nm and 735 nm) have a half-width of about 8 ns, corresponding quite well to the laser pulse width of 8 ns.

The target is a dense tree of Quercus ilex illuminated vertically by reflecting the laser beam with a mirror

Figure 3. Time resolved fluorescence signals as monitored by the transient recorder TDS540 from Tektronics. The pulse length of the excitation is 8.5 ns. The small shift in the maxima is due to different electronical delays in the gate electronics. A mirror used for beam guidance is the source of the additional fluorescence at 440 nm.

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mounted above the tree. The red fluorescence signals indicate that the fluorescence originates from a dense leaf layer without penetrating the tree. In contrast to the red chlorophyll fluorescence, the fluorescence at 520 nm and 440 nm shows a different structure. The blue fluorescence signal has a second peak about 12 ns prior to the main peak, which can be recognized also in the green channel. In addition, both signals have a different decay compared to the chlorophyll fluorescence. Thus it is assumed that an additional fluorescent target was hit. From the time difference between the two peaks of about 12 ns, it can be calculated that the distance between the crown of the tree and the first fluorescence event is about 2 m. Coming back to the setup of the measurement with the mirror about 2 m above the tree, one can conclude that the glass of the mirror partially absorbs the UV-laser light and shows an intense blue fluorescence with a long decay. This example demonstrates the usefulness and the necessity of time-resolved measurements to check the spatial ori- gin of laser-induced fluorescence signals of vegetation.

For airborne operation it is required to determine if the fluorescence comes from vegetated ground or the crown of a tree. Nevertheless, we conclude that the DLidaR-2 has a spatial resolution of = 2 m, mainly limited by the pulse length of the laser.

The data presented in this article are therefore measured without using a mirror. The leaves of Quercus pubescens were illuminated by the excitation spot hori- zontally.

For data processing, the time-resolved signals are analyzed with regard to the maximum of fluorescence.

The maximum fluorescence values are corrected with regard to the gain of the photomultiplier and to the laser pulse energy. These system-corrected data can be correlated with environmental parameters to investigate variations of the plant fluorescence and, as shown above, under special conditions variation of the fluorescence efficiency.

RESULTS

The daily cycle of the laser-induced fluorescence signals at 440 nm, 685 nm, and 730 nm measured on 10 October 1991 is shown in Figure 4. The target, a healthy evergreen oak, Quercus pubescens, is about 30 m away from the fluorescence lidar. The diameter of the laser spot at the target is 30 cm in order to investigate an ensemble of leaves, while the total laser energy is 35 mJ.

Under full sunlight the photon density of the excitation laser beam was 15,000 times higher than the sunlight within the gate width of 50 ns. But related to the time scale of 1 s, the photon density was 1200 times below the highest solar irradiation.

The data presented in Figure 4 are mean fluores-

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I N ch440 ---*,- ch685 --a-- ch730 ... global irr. I

Figure 4. Daily cycle of the fluorescence signals at 440 nm, 685 nm, and 730 nm (left y-axis) of Quercus pubescens, and global irradiation (right y-axis) measured in 10 October 1991.1/teinstein / (m 2 s) is equivalent to 6022 x 1017 photons / (m 2 s).

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cence values averaged over 100 laser shots. This is equivalent to a measuring time of 8.5 s. The standard deviation is less than the dimension of the dots. The corresponding global irradiation monitored parallel during the afternoon of 10 October is presented in the same figure.

From the beginning of the measurement at 1:45 p.m., the global irradiation decreases continuously from 1750 ~ E / (s m2)] while all fluorescence signals increase.

The red fluorescence at 685 nm and 730 nm increase by a factor of about 3.5 and the blue fluorescence at 440 nm increases by a factor of about 2.

During the afternoon of 10 October, measurements of the carbon fixation rate were performed with a porometer (CQP 130, WALZ). The correlation of this physiological parameter with the fluorescence signal is given in Figure 5. The nonlinear dependence demonstrates that the chlorophyll fluorescence increases with decreasing rate of carbon fixation as expected from the models.

The variation of the fluorescence ratios F685 / F730 and F440/F685 are presented in Figure 6. As expected from the previous diagrams and from the models, the fluorescence ratio F685/F730 is nearly independent of ambient light in contrast to the blue-red ratio F440/

F685, which decreases by a factor of 2.2 with decreasing light.

The linear correlation of the blue-red ratio F440 / F685 with the global irradiation confirms the results presented in Figure 6. The linear correlation coefficient is 0.976. The independence of F685 / F730 from ambient light is proved by a linear correlation coefficient of 0.552 coupled with a slope of -0.0005.

From the stress experiments, the branch of Quercus pubescens investigated during 10 October was cut in

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Figure 5. The relation of the laser-induced chlorophyll fluorescence at 685 nm to the carbon fixation rate measured si- multaneous with a Walz CQP130 porometer on Quercus pu- bescens. The inverse relation between the two parameters can easily be seen.

the late evening and mechanically fixed to its original position. Thus it was possible to measure the drought stressed branch of Quercus pubescens on 11 October with the same experimental setup and geometry. In advance of the lidar measurements, the physiological investigations with the porometer and a modified PAM- fluorometer revealed the hypothesis that the cut branch of the oak has developed drought stress symptoms during the night. The carbon fixation rate was zero, the Genty parameter (see, e.g., Genty et al., 1989; Seaton and Walker, 1990 for definition) of the stressed leaves was reduced to about 0.15 and the Rfd value (see, e.g., Lichtenthaler and Rinderle, 1988 for definition of Rfd value) was also reduced.

In Figure 7 the daily cycle of the fluorescence signals at 440 nm, 685 nm, and 730 nm of the drought Figure 6. Daily cycle of the fluorescence ratios F685/F730 and F440/F685, measured on 10 October 1991 at a healthy oak, Quercus pubescens.

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Figure 7. Daily cycle of the fluorescence intensities at 440 nm, 685 nm, and 730 nm (left y-axis), and global irradiation (right y-axis) measured on 10 October 1991 at a drought stressed branch of oak, Quercus pubescens.

stressed branch of Quercus pubescens are shown as well as the variation of the global irradiation from the morning till noon. On 11 October, the weather and thus the global irradialSon were very variable compared to the day before, as can be seen the same figure. In the early morning it was only partially overcast, but cloudiness increased, resulting in rain in the afternoon. Thus the measurements were stopped at noon. Nevertheless, a different trend of the fluorescence signals with decreasing ambient light is observed. While on 10 October the fluorescence of a healthy oak increases with decreasing global irradiation, the fluorescence of the drought- stressed oak decreases with decreasing light. In addition, it is remarkable that the absolute fluorescence intensity of the stressed branch is strongly reduced in comparison to the healthy. This is clearly seen by the different factors of the two y-axes. This fact is verified by the PAM measurements which also showed a strongly reduced signal of the stressed leaves compared to the healthy ones. On the other hand, the results show that the average fluorescence ratio F685/F730 is 0.3 for the stressed branch, which is comparable with the value for the nonstressed branch (0.35).

CONCLUSION

The daily cycle data of laser-induced fluorescence of a healthy and a drought stressed oak (Quercus pubescens) demonstrate for the first time that it is possible to monitor the physiological state of plants in the far field in single shot operation.

It is also shown that the chlorophyll fluorescence ratio F685/F730 is independent of global irradiation.

The influence of drought stress on F685/F730 can be neglected if an error of about 15% is assumed. This

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result is consistent with the simple leaf model of Dahn et al. (1992) and with the experimental results of Hak et al. (1990) obtained in the near field. Both groups found that the chlorophyll fluorescence ratio /7685/

F730 mainly depends on the chlorophyll concentration and only to a minor degree on the fluorescence efficiency. Thus, if one regards the physiological state of plants on different time scales, one can conclude that the chlorophyll concentration determines the physiological state on a long-time scale (growth rate) and the fluorescence efficiency on the short-time scale (variation of the global irradiation).

The inverse dependence of the steady state chlorophyll fluorescence, F685 or F730, with the carbon fixation rate demonstrates that the fluorescence intensity is tightly connected to photosynthesis. The data presented here confirm that the laser-induced spectral resolved fluorescence measurements in the far field give information about the physiological state of plants and that a parameter equivalent to the carbon fixation rate can be remotely monitored.

If drought stress becomes visual, a strongly reduced fluorescence intensity at 685 nm and 730 nm is observed (see also Theisen, 1988). But to identify drought stress, it is necessary to determine the chlorophyll concentration by the fluorescence ratio F685/F730. This result is slightly in contrast to the general interpretation that a disturbed photosynthetic apparatus increases the chlorophyll fluorescence (Renger, 1982).

The increase of blue fluorescence at the wavelength 440 nm with decreasing global irradiation and therefore with decreasing photosynthetic activity of the healthy plant Quercus pubescens contrasts the findings of Chap- pelle et al. (1991) while the blue fluorescence of the drought-stressed branch at the same tree shows a de- crease of fluorescence with decreasing global irradiation, which is in agreement to Chappelle et al. (1991).

We thank our colleagues of the University of Tuscia for the organization of the LASFLEUR campaign in October 1991 and their good cooperation. In addition, we thank all colleagues working in the LASFLEUR project for their critical and fruitful discussions and for their measurements helping to understand

•lant fluorescence in more detail. The LASFLEUR project (EU 380) is sponsored by grants of the BMFT under Contract No.

033 9290 A6.

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Remote Sensing Vegetation Status by Laser-Induced Fluorescence