Laser Induced Leaf Fluorescence A Tool for Vegetation Status- and Stress- Monitoring and Optical Aided Agriculture

Laser Induced Leaf Fluorescence

A Tool for Vegetation Status- and Stress- Monitoring and Optical Aided Agriculture

Wilhelm Luedeker, Kurt P. Guenther, Hans-Guenter Dahn Institute of Optoelectronics, Optical Remote Sensing Division

P. 0. Box 1 1 16, D 82230 Wessling

Wilhelm.Luedeker@dlr.de, Tel.: +49 8 153 28-1 339, Fax.: +49 8 153 28-1444

Abstract

Sincethe second half of the 1980'th several efforts started to establish the laser induced vegetation fluorescence as remote sensing tool to detect the growth and I or stress status of plants. The most extended European project, the EUREKA project LASFLEUR (1989-1994), demonstrated the technical feasibility and the significance of the sensed data.

Exciting leaves with strong light pulses anywhere in the UV-A region of the electromagnetic spectrum stimulates a broad fluorescence emission from 400 to 750nm. This emission is separated in two main components, the ,,blue-green" (400-600nm) and the red fluorescence region (680-750nm).

The blue-green band is originated by polyphenolic compounds of the cell walls, NADPH of the photosynthetic apparatus and possibly by several other plant pigments, except chlorophyll, which is the only emitter of the fluorescence at two bands in the red and in the NIR respectively. On the basis of the photon flux in these channels and with additional information, derived from eg. the elastic back scattered signal, the time duration of back scatter and fluorescence signal, environmental light conditions etc. a large set of vegetation parameters could be determined.

During several demonstration campaigns status parameter as eg. the chlorophyll concentration, photosynthetical activity and canopy structure were investigated. Additionally stress conditions as eg.

drought-, UV- stress and infection with different kinds of fungi were examined as well as the differentiation of plant types as e.g. mono- and dicotyledons.

Extrapolating the knowledge of the EUREKA project leads to two different main applications. First with an advanced airborne remote sensing system monitoring of the vegetation status and stress conditions may be possible independently of other remote sensing techniques or the data may be used as input parameter for eg. passive radiometer images. The second application will be a miniaturized sensor for agricultural machines giving direct access to plant parameter and hence the possibility for individual plant treatment as eg. determining the growth state, fertilization or weed protection.

Keywords : plant morphology I pigmentation, photosynthesis, blue fluorescence, chlorophyll f luorescence, annihilation effect, lidar technique.

Introduction

Beginning in 1989 the EUREKA project LASFLEUR (EU380) started as a combined effort of research groups from France, Italy, Sweden, the EEC and Germany in order to demonstrate the

feasibility of monitoring the physiological status of

plants and canopies by laser-induced fluorescence (see special issue on fluorescence measurements of vegetation of Remote Sensing of Environment, 1994).

During the following six years a huge amount of experience was collected in terms of remotely sensable plant parameters as e.g. plant types, pigment concentration, photosynthetic activity and plant stress conditions.

At this development level the fundamental mechanisms of the leaf fluorescence, the measurement opportunities, applications and future practical operations should be pointed out by this publication.

Theory of leaf fluorescence

The absorbed photosynthetic active radiation (PAR) of the solar irradiation has three paths of dissipation in a plant. The most important destination is the conversion in the photosynthesis of plants, stored as chemical energy, directly linked with the uptake of carbon dioxide and thus linked with the primary productivity. Two other paths are remaining to keep plants total energy balanced, the emission of thermal energy and the emission as fluorescence light.

The thermal energy budged is filled up with solar energy from the visible (VIS) and the short wave infrared (SWIR) range of the solar spectrum. SWIR radiation is directly absorbed by the leaf internal water content. The VIS range contributes via the exciton transfer inside the antenna pigment of the reaction centers (PS I; PS II) and light harvesting complex (LHCP). In this process the absorbed photon energy is transformed to energy quantities required by PS I and II. The surplus of energy is stored in oscillating and rotation energy levels and thus finally converted into heat.

At the PS I and II the matching energy quantities are dissipated in the so called light reaction, in further convolution as heat, or finally in emission as fluorescence light. The conversion probabilities for the heat path and the fluorescence are constant in time, whereas the conversion rate at the light

reaction is a function of the state of the reaction center (electron transfer chain) and the

phosphorylation state of the photosynthetic active cell membranes. Equation (1) describes the fraction of sun excited fluorescence light (F(t)) which is emitted by the reaction centers:

F ( t ) =

^kFl

* I d2

⁽¹⁾

k

Fluorescence

+ k + k

Hear Phorosvnrhess^•

_{. (0 M)}

PAR ^Abs-Sun

k : conversion probability 4^: state of the reaction center M : phosphorylation of membrane 'Abs-Sun : absorbed spectral irradiance

From this formula it can be seen, that the behavior of the fluorescence in time gives access to the relative changes of the photosynthetic activity if one assumes that "4"and"M" are depending on the time. A similar formula can be given for the fraction of heat conversion which would describe the

variations in the photo acoustic spectroscopy of leaves (Buschmann and Grumbach 1985).

In the laser inducedfluorescence (LIF) with LIDAR-technique a "high-power" laser pulse "Abs-Laser"

is added to the absorbed sun irradiation "abs-Sun" and induces a strong fluorescence pulse, which exceeds the normal sun induced fluorescence in a way that Fun(t) is just a small fraction of the total

fluorescence signal in comparison to FLavej-(t), at least for the duration of the excitation pulse.

With adequate technical set-up this signal can be separated from the passive reflectance spectrum even under daylight conditions at distances, ranging from direct contact to several hundred meters.

NADPH

Figure 1 : Z—Scheme of the electron transfer chain with corresponding transfer times of distinct stages (Keller 1986). The letters indicate different involved molecules and protein complexes, their vertical position indicates the relative redox potential of each state.

The now arising fundamental question is : What is the status of a reaction center and what does the excitation light pulse induce in it?

kphotnrhevis(ø ,M)describes the instantaneous ability of transferring light energy to the photosynthetic apparatus. If the photosynthesis runs efficiently "kph(,)" is high and thus F(t) is reduced and vice versa. An efficient photosynthesis means that all involved processes, the electron transfer chain (ETC) (fig. 1) and the linked "Calvin cycle" are running well.

For dark adapted leaves the reaction centers PS I and II are in ground state (oxidized; not excited), the so called plastochinone pool PQ (slowest component in the ETC; fig. 1 .)isempty, the Calvin cycle is not working and the membrane of the grana stack in the chloroplast is not energized.

Slight light quantities (h*D) at this phase induce an excitation of the PS to PS. Due to the very fast process of electron transfer (ips) from PS to the primary electron acceptor A1 this process is very efficient (kph0I.=>high) and thus the F(t) is very low. In the commonly used pulse-amplitude- modulation fluorometrie (PAM) (Schreiber et al. 1986) this situation indicates the so called F0 fluorescence. The intensity of the excitation (measurement) light is as weak that no photosynthetic activity is stimulated.

Illumination of a dark adapted leaf with an intense flash of several milli- seconds up to one second duration is flooding the PS's with energy and thus the ETC with electrons (PS are reduced). But these short pulses are to short to trigger energy dissipation by chemical processes. Thus all fastprocesses are energized (all reaction centers are closed) but the energetization of the membrane is not jet

initialized. kpht(, 5 dropping down and F(t) is reaching the absolute maximum value. This

fluorescence is called (Faxjum) 111thePAM fluorometrie.

A continuous illumination with non saturating light (so called actinic light) induces photosynthetic activity and causes electron flow in the ETC. After several seconds until minutes of illumination all contributing processes are in equilibrium with the supplied light and thus the fluorescence has reached a steady state value F. The transient of the fluorescence during illumination of dark adapted leaves is called Kautzky —Effect (fig.2.) and the fraction of steady state fluorescence compared to the maximum fluorescence (RFD-value) (Lichthenthaler and Rinderle 1988 )isassumed to be an indicator of the physiological ability of a plant.

CID

00

TI) TI)

E :i.

"spillover"

time [SI

Figure 2. : Kautzky kinetic of a cucumber plant measured with the DLidaR-2. The detected fluorescence at 685 nm is exclusively induced by the laser pulses. Illumination with a 500 W

halogen spot light only influence the

photosynthetic state and thus kph0r0.. Its

contribution to the fluorescence signal,

especially as excitation source, is negligible.

global irradiance [W*m2]

Figure 3: The inverse relation of F 685 of

Quercus Pubescens (measured with DlidaR-2

from remote) with steady state global

irradiation 10thOctober, 1991.

Depending on the plant type the photosynthetic activity is proportional to the absorbed irradiation and thus F, is a function of the irradiation (

I )

Thisrelation is only valid if the plants have time enough to adapt their metabolism to changes in the light environment, as it could be seen from plots of fluorescence changes with global irradiation under steady state (fig.3.) and transient light conditions (fig. 4.).

July 3rd^wasa cloudy day with frequently changing phases of high illumination and cloud cover. A correlation of F685 and global irradiation could not be found as seen in fig. 3. But there are two

examples (fig. 4.) where the fluorescence is

tracked over a longer time period. The gray data points sign fluorescence values in time where the illumination rises from medium to a high level

(450 —940Wm2) and remains at this level until its dropping down again to 350 Wrri2. The black dots

indicate the opposite direction, changes from

medium to low light. The general behavior is in full accordance with the experience from other experiments (fluorescence depression at high light

(.0 U..

0 30 60 90 120 150 180

6.0

(0U- 4.0

2.0

0.0

level and vice versa),.and the mentioned theory of energy dissipation in a leaf.

The described link of the fluorescence intensity to the physiological state of a plant is complemented by the spectral features of the whole emission spectrum from 400 to 750 nm, which is coupled in

8.0 several

ways to the plant morphology, as e.g.

pigment constituents and pigment concentration or indicates a possibly appearing leaf infection.

The fluorescence exhibits (fig. 5.) two dominant emission

bands, 400 —

⁶⁰⁰ (blue-green

fluorescence BG) and 650 —

750

nm (red

fluorescence; F685, F730).

From experiments it is known, that the emissions

at 685 and 730 nm are both linked to the

photosynthetic system as described before and thus

0 100 2W 300 400 500 600 showthe same variations in time. In contrast the fluorescence ratio F685 I F730 of an individual plant is constant in time and depends only on the optical properties of the leaf (Dahn et al. 1992).

The fluorescence emission and the pigment

absorption bands are overlapping around 685 nm,

hence this emission is reabsorbed selectively

4.0

3.5

3.0 L()

(0 25

LL 2.0

1.5

100 300 500 700

global irraclance [W m1

Figure 4: Changes of F685 of Picea Omorica with global irradiation for a cloudy day (July 3rd 1992)

wavelength [ nm I

L

C')C

ci)

C

Figure 5.:Fluorescenceemission spectrum of a maize plant grown in the greenhouse. F685 and F730 originates exclusively from the leaf internal chlorophyll. The _blue-green fluorescence (BG) is emitted primarily by phenolic components of the cell walls.

during its path though the leaf tissue. The result is an exponential dependence of the ratio F685/F730

from the parameter : mean free light traveling length in a leaf, scattering coefficient and

chlorophyll concentration.

The only time dependent variation found in the ratio occured during the transitions form fully dark adapted plants to light adaptation even at low light quantities (e.g. early morning, evening, during Kautzky Kinetic) and vice versa. Under day light no significant

dependence or

correlation

900 respectively

of the ratio and global irradiation

could be found. There fore we assume that these changes are related to variations in the optical

properties of the leaf tissue. A potential

mechanism could be the orientation of the plant organells (e.g. chloroplasts) towards the arising

illumination, but this is matter of further

investigations.

Nevertheless, the ratio gives access to measure relative variations of the chlorophyll concentration

for a plant species if one assumes a similar

morphology for the individual plants. This mean that the leaf internal scattering coefficient and the leaf geometry are comparable.

The origin of the BG fluorescence is more difficult

to identify and is still matter of scientific

800 discussion. Fig. 6 shows categorized images of a

wheat leaf cross section, recorded with a

fluorescence microscope.

From this images it can easily be seen that the blue-green fluorescence originates from the cell

walls and only a weak fraction is emitted by structures from deeper cell layers. No blue

fluorescence of the chloroplasts is evident because the red chlorophyll fluorescence is the dominant factor. Nevertheless it is known (Cerivic et al.

U

0.0

400 450 500 550 600 650 700 750

. •$

_'

A:

.'

.'

L

Figure 6. : Leaf (wheat) cross section under fluorescence microscope, categorized in terms of chlorophyll fluorescence (left) and blue fluorescence (right). SOLID LINE . leafenvelope; Left image : DARK =chloroplastfluorescence;GRAY =fluorescencefrom deeper cell layer or damaged chloroplasts; Right image : DARK = ^bluefluorescence of cell walls; GRAY =fluorescence

of

deeper layer or vacuole. Image recorded by: Stober 1993; categorized by DLR.

1993) that NADPH in the chioroplasts is emitting blue fluorescence. Also on the cell level it is shown that fluorescent coenzyms such as NADH or NAD(P)H are very sensitive bioindicators of metabolic functions such as the degradation of glucose or respiration(Schneckenburger and Koenig 1992). Thus the blue NADPH emission depends on the physiological state of the plant.

For leaves and with an excitation wavelength below 400 nm this effect is completely covered by emission of the cell wall, emanated by several pant constituents. Stober and Lichtenthaler (1993) enumerate plant phenolics, ferulic-, chiorogenic- and caffeic acids, as well as cumarins as source of the blue emission and alkaloids and flavonols as source of the green fluorescence.

An additional source of BG fluorescence may be the coverage of the leaf surface by other organic materials as e.g. with fungal infections (Luedeker et. al. 1996). The fluorescence emission spectrum of the infected leaf is affected in two different ways.

-

_the auto fluorescence of the fungi increases (or changes) the BG fluorescence selectively and

-

_the surface layer lowers the red fluorescence by absorbing the excitation light and therefore decreasing the penetration depth. The same effect is seen if the excitation light is diffusely reflected by an additional tissue layer at the plant surface.

The latter behavior is also known from UV protection pigments within the epidemal cell vacuoles (Schnitzler et al. 1996) whitch hinders the "UV" excitation to penetrate deeper cell layers and thus depresses the chlorophyll fluorescence selectively. Usually these pigments (e.g anthocyanin) are solely absorbers and do not contribute to the total fluorescence signal.

Technical

_aspects

Fromthe theory the constraints for remote detection of the fluorescence signal are easily determined.

-

_The exciting light pulse should be weak or fast enough to keep the plant in an unchanged physiological state

-

_and should be strong enough to induce fluorescence which exceeds the passive reflected and sun induced fluorescence radiance.

The basic parameter which describes these constraints is the signal —to^— _background ratio (SBR) given by equation 2.

SBR= ^'-'

₂

R+F.

sun s—i

⁽⁾

with : SBR :=

Signal-to-Background-Ratio := passivereflected radiation

F1 :=

^laserinduced fluorescence := suninduced fluorescence

-

_Whenthe SBR is less than "1.0" single shot measurements are not possible and other techniques as e.g. lock-in technique have to be applied to increase the SBR.

- If

SBR 1 .0 the background has to be measured separately and subtracted from the fluorescence signal.

- If

SBR >> ¹.0 the background can be neglected and single shot measurement of the fluorescence is possible. In order to achieve a SBR >> ¹ one can increase the excitation intensity when the spot

diameter should be fixed or decrease the spot diameter as far as necessary to reduce the

background photons.

But an another limiting factor, the "annihilation effect" (Campillo et at. 1976), has to be regarded.

The annihilation effect is attributed to the nonlinear dependence of chlorophyll fluorescence with increasing excitation light.

If the photon density of the excitation pulse exceeds

i013 [photons*cm2*2Opsh] at the photosynthetic system Mauzerall (1975) proposes that exciton — excitoninteractions in the antenna pigments or light harvesting protein complexes are responsible for this effect. Campillo et al. (1976) supposed singlet —_singlet annihilation at the reaction centers as possible source of quenching. Nevertheless for both explanations the population of excited states is to high and thus the absorbed energy is lost by an intrinsic mechanism and suppresses the quantum yield of the fluorescence, indicating ostensible changes in the photosynthetic state. In terms of plant irradiance this upper limit means that dependent on the excitation wavelength the peak power is

limited to roughly 150 —300[kW*cm2] without annihilation effect.

For the detection system of a fluorescence lidar the following characteristics should be fulfilled:

-

_highsensitivity and

-

low noise to sense also weak light levels,

-

_fastrise time to detect the ns pulses,

-

_highdynamic range with radiometric linearity and

-

_widelyvariable amplification.

A photo multiplier tube (PMT) satisfies these requirements except the dynamic range because the high sensitivity causes a high (but limited) dynode current already at low continuous illumination levels. To overcome this problem the PMT is switched on just for the duration of the expected fluorescence signal and switched off for the rest of the time.

The analog output of the PMT is fed into a transient recorder, sampled by a gated integrator or converted to a voltage proportional to the peak signal. Finally an analog to digital converter is used

and the data are stored on a computer.

The DLidaR-2, developed during the LASFLEUR project, uses a tripled Nd:YAG laser with pulse duration of 8 ns and pulse energy of typical 35 mJ limiting the spot diameter to 4.5 cm. From these data it is clear that the operation distance is limited for a given excitation pulse peak power and a defined SBR depending on the expected fluorescence quantum yield and an additional term which characterizes the path radiance between sensor and target.

For near field measurements the background light can be discriminated by simply decreasing the spot to a "point". The necessary excitation power to induce sufficient fluorescence photons is orders of magnitude lower as for far field operations and thus the risk of annihilations effects prevented.

The optical concept is in this context of lower interest, because the principal measurement method is mainly not affected by it's design. During the LASFLEUR project several approaches have been realized For more information see reference GUnther and Schmuck 1993, Luedeker 1995.

Applications

Inthe following section several applications of the DLidaR-2 are presented, giving an overview which plant or canopy parameters can be measured with a ground based fluorescence lidar. Most remote measurements were performed under field conditions.

Drought stress

Quercus Pubescens is well known as a plant which

responses very fast to water deficiency. In a two day lasting field

⁰⁷⁵

experiment

the laser induced fluorescence ;

response

of a Quercus Pubescens to normal .

_0.50

conditions

and water deficiency was monitored. It was shown that the inverse

correlation of chlorophyll fluorescence with ^0.25 globalirradiation is changed under drought

stress (see fig.7). To suppress the water

_'

supplyof the branch of interest it was cut at _{200 300 400 500 600}

the evening, repositioned and fixed in order globalirradiance [VV*m2]

to get the canopy structure as the day before. . . .

. . . . Ficr. 7: Global irradiation versus F685 for Quercus

The increase of F685 with increasing global

b

. . . . . . Pubescenswith cut branch on a sunny day.

irradiance is typical for water deficient

plants and blocked photosynthetic activity. It

is just in opposite to healthy plants. The interrupted water supply prevents electron flow in the ETC.

6.5 Pigment

concentration

data for fit

In

October 1994 a field campaicn was

'

^{5 5}_. — nonlinearfit

ft ^performed to investigate the vitality of Scots

pine forests at two test sites in eastern

4.5 Germany. One is located in Neubrandenburg

(northern part) with _relatively clean

3.5

t

environmentalconditions. The other location

t

^isclose to Bitterfeld in a region where brown

2.5

t

coal was used for industrial purposes until

1990. This area was highly loaded with 502,

1.75 1.85 1.95 2.05 2.15 225 2.35 245 ^.

. .

NO

and ashes with a now slowly decreasing

Chlorophyllconcentration [mg I g (dry we t)]

pollution.Therefore a typical set of damaged tree is present.

Fig. 8: Correlation of fluorescence ratio F685/F730 The measurements have been carried out at versus chlorophyll concentration. R2=O.82; three five individual trees of three different groups ratios correspond to one pigment concentration within three (reference) and five (polluted)

test sites.

The pigment measurements

differentiate test sites, whereas the

fluorescence measurements are averaged for each tree group. Figure 8 shows the non linear

dependence of the fluorescence ratio F685/F730 and the chlorophyll concentration of pine needles. A correlation coefficient of 0.82 was calculated for this plot. The variation in this figure is mainly caused by natural variability of the plants and thus three ratios correspond to one pigment value. The pigment concentration in the polluted region is found to be higher than in the reference area (Schulz et al. 1996).

1.00

Plant distinction and UV -

_protection

[O9.June

1Z14.July 1994 1994

I

^T

^a

^CJ

^a

^C')

^a I

plant type

Fig. 9: Fluorescence ratio F4401F730

^for

dicotyledone (ainare (Dl), calystegia sepiuin

(D2), abutilon (D3)) plants and monocotyledone (alopecurus pratensis (Ml), lolium multiflorum (M2), avena fatua (M3)) plants two weeks old (left bar) and fife weeks old (right bar). Excitation wavelength ex^{= 355}nm

laboratory

open sky

JIiIi

plant type

Fig. 10: Fluorescence ratio F440/F730 for the same plant set grown under UV-protection (left bar) and open sky conditions (right bar).

representative samples of each plant species have been investigated. The measurements started with two weeks old sprouts of different plants

grown under greenhouse conditions. The

experiments were carried out under constant light conditions.

The results (fig. 9.) confirm the supposed

features of the emission spectrum of sprouts (left bar). The effect is enhanced after additional five weeks of growing under greenhouse conditions

,

(rightbar). The reason for this typical difference

is not yet identified. To make an advanced

separation among plants types or even species feasible in the future, systematic investigations of

the fluorescence emission spectra and the

corresponding plant constituents are necessary.

As already mentioned plants may synthesize UV

— protecting pigments depending on the light quality during their development. Thus the two week old sprouts have been divided in two sub- samples to investigate this effect. One sample was growing under field conditions with direct sun exposition and the other just one meter apart from the first, but UV —_protected

by a glass

window (cut-off wavelength: 400 nm) for three weeks. During this period the weather conditions were very good with high UV —^treatment. The result (fig. 1 0) is again a distinction between the

two plant types even if the effect is not as clear as for the UV —_protectedplants. The lowest ratio for the "M" —_plants grown with sun exposition exceeds the highest "D" — _plant value roughly 40%.An increase of the ratio by a factor above

"15 - 19" for all D-plants (2.8 —6.3, M-plants) shows the high efficiency of the UV —_protection mechanism. It is caused primary by absorbing

pigments in the epidermal leaf layer which

hinders the penetration of the UV excitation light (355 nm) into the cell tissue. These pigments do From many experiments it was found that different plant types exhibit different spectral fluorescence emissions, especially in comparison of the blue and red fluorescence if they are excited with a tripled Nd:YAG laser (355 nm). It was supposed Chappelle et al. (1984) that monocotyledone plants emanate

a more pronounced blue fluorescence than dicotyledone types. To verify this findings three

8

0

Co

U-N-

0

U-

6 4 2 0

CO

24

0

20

c) 16

.'— C'.I ()

not contribute remarkable to the blue fluorescence. Thus the strong variation with UV —treatment originates selectively from the reduced chlorophyll fluorescence signal.

1.8

___________

N1 S11ffIN2 H

I •. _channel

^440/520 combination

ii

^{685/730 I}

Fig.1 1 : Spectral feature of pine needles from

reference (N) and stressed (S) test site

separated for one and two years old needles (1) / (2), measured at non necrotic needled center.

Necrosis

Necrosis is a widely spread symptom of plant response to environmental stress present during our

field campaign in October 1994 (see: pigment concentration). The data (F440/F520) of this

investigations showed a very high correlation with the necrosis index (R=O.91). The "needle damage index" describes a visual estimation of the fraction of necrotic needles per total needle number, regarding the progress of the necrosis.

The high correlation coefficient resulted from two clusters for each test site without any data points between. Thus we decided to support the field data with measurements in the laboratory on single

0 1.0

C) C.)C

C) C.) Cl) C)

0 'I-

1

.

0

C) C.)C 0)

C.) Cl)

0

'I-

i

N 1 S1llN2S2

liii hi

440/520 685/730

channel

combination

Fig.

I 2: Spectral feature of the same pine

needles as fig. I I but measured at the necrotic needle tips.

needles harvested at the same test sites to investigate the spectral features of the necrosis with a spectral fluorometer in comparison to healthy plant material.

On the needle center without visible necrotic cells, no significant change in the F440/F520 ratio was observed (fig.1 1). The ratio F685/F730 showed also no indication for plant damage at the needle center. In accordance to the pigment measurements the needles at the test site "5" contain more chlorophyll than the "N" needles. The investigations at the needle tips where the necroses typically arises showed completely different results. The green fluorescence emission is significantly increased accompanied by a reduction of the chlorophyll content which is visible in an increased F685/F730 ratio for the second needle age group. Figure 1 2 demonstrates that necrotic needles also occur at test

site "N" but not with the same high significance.

Unfortunately the fluorescence feature (F440/F520) directly in the necrotic zones contradicts the results of the lidar measurements. The laboratory measurements showed an decreasing ratio whereas the field data showed a clear increase.

This behavior is explained by needles of the 3rd_agegroup which were still on the trees in test site "S"

but with a completely different morphology (e.g. no chlorophyll). Thus the lidar measurements, with an excitation spot size of 25 cm in diameter, are regarding all needle (even dead needle) material, whereas for the physiology measurements they are completely neglected. It was concluded that the

spot size was too large to detect necroses.

An active remote sensing system for vegetation monitoring will be able to detect the vegetation status in terms of photosynthetic activity, stress conditions, chlorophyll concentration, canopy structure and

some types of plant infections. This technique allows the detection of symptoms as e.g. changes in the fluorescence activity in case of short term (often reversible) stress or via the pigmentation or canopy

structure in case of long term stress.

Discussion of remote detection set-ups

Farfield lidar

Beside the requirements to the electronics mentioned before an active far field lidar set-up requires a light source with high peak power and short pulse duration. The DLidaR-2 of the DLR uses a tripled Nd:YAG laser with Prnax 4.4 MW, Xex 355^nmand 'tex 8 fl5 pulse length. As demonstrated in the

chapter "applications" the UV —_protectionof plants can prevent the chlorophyll excitation very efficiently and thus reduce the maximum operating distance for a given peak power. To overcome this problem the wavelength has to be shifted towards the visible part of the spectrum. Taking into account eye safety restriction it is supposed to set the excitation wavelength just below 400 nm. With a laser like this it would also be guaranteed that the blue fluorescence will be excited efficiently.

An airborne system needs a light source with high repetition rate to make area or at least profiling investigations feasible.

There is no commercially available laser systems that meets all requirements (Pmax, 2ex, Cex, repetition rate) at the moment. Diode pumped solid state laser technology seems to be the most promising laser development to meet the requirements in the future. From our point of view the 3rd or 4th harmonic of a Nd:YAG laser in combination with frequency mixing as well as a frequency doubled Cr:LiSAF or Cr:LiCAF laser could be realized within a short or medium time schedule. But the actual cost driving factor is the price for the laser diodes as pump source for the solid state laser.

Independent of the optical set-up the timing-, detection-, and signal processing electronic requires precision electronics resulting also in expansive hardware costs. Thus a commercial application seem not to be realistic.

Nearfield lidar

Near field operations require less expensive components. The pulse power can be reduced due to the background discrimination by a reduction of the excitation spot to a "point". The PMT could be operated with a continuous gain voltage because the photon (electron) flux in the opto-electronic system is reduced. Variable adjustment of transmitter and receiver timing is not necessary because the fluctuation of the delay time is negligible in comparison to the pulse length.

First laboratory tests with a near-field fluorescence lidar demonstrate the feasibility of

miniaturization. As excitation source a commercial available pulsed laser diode (CRYSTAL PDL 670) (Pex 500 mW, ex 670 nm, 'rex I 8 ns, repetition rate up to 10 kHz) was used. The detector was a compact PMT (HAMAMATSU H5783) in continuous mode. The fluorescence signal was separated from the elastic back scatter signal by an interference filter (center wavelength = 692 nm, aperture = 1 0 mm) mounted directly at the entrance aperture of the PMT. The fluorescence photons emitted from a leaf were collected by a glass lens. The measurement distance for this optical set-up was 40 cm under laboratory light conditions.

From these data it is estimated that an optimized optical system will allow measurement over distances of lm and above, with adequate signal-to-background ratio for detecting chlorophyll fluorescence (e.g. pigment concentration, activity or even vegetation recognition).

The laser diode seems to be the best available light source for commercial purposes because it fulfills

all prerequisites for a non actinic excitation source, it is fast and can be operated in a rough

environment as it occurs e.g. in horticultural or agricultural applications.

With the availability of blue or UV diode laser with similar specifications the same set-up can be used to investigate additionally the blue fluorescence.

Conclusions

•

The investigation of vegetation with laser induced fluorescence by means of remote sensing is a helpful tool to determine the status of plant in terms of photosynthetic activity, physiological

status and vegetation structure.

.

The technical requirements for detection of a defined plant parameter set are sufficiently determined. The realization allows advanced investigation with the

- farfield set-up, but limited to scientific or governmental purposes or

- specialized purposes with a relatively simple near field set-up which give access to many commercial applications.

S The far field lidar technique could be improved by the development of a rugged and easy to handle solid state laser system, also adequate for airborne operations.

.

Theapplication spectrum for the near field system could also be improved by the development of a comparable light source in the blue or UV region of the spectrum as it is available with the power enhanced and pulsed laser diode at 670 nm.

.

Bothtechniques require expensive high power diode lasers, one as optical pump for the solid state laser, the other as excitation light source for the fluorescence. This new application field could

induce a growing commercial market for these diodes.

.

The utilization of a near-field lidar in the horti- or agriculture as on-line method allows the regulation of e.g. nutrients via the detection of the chlorophyll concentration.

Acknowledgement

We thank our colleagues of the environmental research center (UFZ) Leipzig-Halle, Section of Chemical Ecotoxicology, for the cooperation and support during the field campaign October 1994.

The LASFLEUR project (EU 380) is sponsored by grants of the BMFT under the contract number 033 9290 A6.

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