ОПТИКА И СПЕКТРОСКОПИЯ, 2Ü12, том 112, № 2, с. 299-3Ü7


УДК 535.8


© 2012 A. Lavrov***, A. B. Utkin***, J. M. da Silva***, Rui Vilar******,

N. M. Santos*, B. Alves*

*INOV-Inesc-Inovagäo, Lisbon, 1000—029, Portugal **Institute of Material and Surface Science and Engineering, Lisbon, 1049—001 Portugal *** University of Lisbon, Faculty of Science, Department of Plant Biology and BioFIG, Lisbon, 1749—016 Portugal **** Instituto Superior Técnico, Department of Chemical and Biological Engineering, Lisbon, 1049—001 Portugal

E-mail: andreiutkin@inov.pt Received July 13, 2011; in final form, September 15, 2011

Abstract—The aim of the present work was to develop a method for the remote assessment of the impact of fire and drought stress on Mediterranean forest species such as the cork oak (Quercus suber) and maritime pine (Pinuspinaster). The proposed method is based on laser induced fluorescence (LIF): chlorophyll fluorescence is remotely excited by frequency-doubled YAG:Nd laser radiation pulses and collected and analysed using a telescope and a gated high sensitivity spectrometer. The plant health criterion used is based on the 1/I740 ratio value, calculated from the fluorescence spectra. The method was benchmarked by comparing the results achieved with those obtained by conventional, continuous excitation fluorometric method and water loss gravimetric measurements. The results obtained with both methods show a strong correlation between them and with the weight-loss measurements, showing that the proposed method is suitable for fire and drought impact assessment on these two species.


Optical methods present considerable advantages for in vivo and in vitro assessment of the physiological condition of live tissues as compared to chemical and physicochemical methods, because they are much faster, non-invasive and non-destructive (see, for example, Berberan-Santos et al. [1—3] and references therein). Laser induced fluorescence (LIF) analysis is particularly interesting for this application because the measurements can be carried out remotely, allowing, for example, difficult-to-access canopies to be inspected. Large plantations and woods can also be efficiently inspected, by scanning an instrument placed at a high viewpoint or by mounting the LIF-LIDAR instrument in an aircraft.

Early research [4, 5] showed that the LIF spectra of plant leaves present two maxima — a local maximum at 685 nm (I685) and an absolute maximum at 740 nm (I740) — the relative intensities of these maxima changing with the physiological condition of the plant pho-tosynthetic system. LIF was applied for estimating the maturity of lettuce [6], differentiating plant species [7—9], assessing potassium deficiency related stress [10], estimating the overall metabolic activity of plants during a defined period of time [5, 11], and studying the influence of ambient light [8], intense UV radiation [4], atmospheric [12] and soil pollutants [12, 13], and excess of ammonium nitrate [14] on plant physiology.

Presently, the most important optical methods for evaluating the plants physiological condition are based on the Kautsky effect (also known as fluorescence induction). In 1931, Kautsky and Hirsh [15] discovered that the intensity of fluorescent radiation emitted by leaves suddenly illuminated after a period of darkness increases from an initial level F0 (usually measured ~20 ^s after excitation) to a maximum value Fm, observed about 1 s later. Kitajima and Butler [16] showed that the maximum potential quantum yield of PS II photosynthesis system is characterised by the dimen-sionless parameter Fv /Fm = (Fm - F0 ) /Fm. A value of

Fv/Fm of 0.832 ± 0.004 was found for healthy leaves of a very wide variety of species [17], while stress due to disease or environmental conditions is indicated by lower values. F0 also depends on the chlorophyll concentration in the leaves [18], which, in turn, depends on the physiological condition of the photosynthetic system, but severe stress may change the basal fluorescence yield, affecting the relation between F0 and chlorophyll concentration. A significant increase of F0 due to heat stress, independent of the chlorophyll concentration, was reported by Havaux and Strasser [19]. The increase of the basal fluorescence intensity probably reflects a disturbance on the organization of thy-lakoid membranes [20].

In vivo chloroplast fluorescence analysis is currently a key tool in photosynthesis research [21]. The Kautsky effect is on the basis of the pulse amplitude

Fig. 1. Layout of the LIF-LIDAR system.


Nd:YAG laser, 532 nm


Ocean optics spectrometer

Fluorescence radiation

Optical fiber

Light gathering optics

Control and data acquisition

modulation method (PAM) [22], which is nowadays the most common plant fluorescence analysis method. PAM was successfully applied to a wide range of plants, including the olive tree, rosemary and lavender [23], Paspalum dilatatum [24], Phillyrea angustifolia [25], the grass Setaria sphacelata [26] and other C4 turfgrasses [27], maize [28, 29], and Arabidopsis thaliana [30], among others. Plant Efficiency Analysis (PEA), which is also based on Kautsky effect, employs continuous wave excitation and measures the rapid rise of fluorescence intensity after a dark-light transition [31]. It provides useful complementary information to the PAM method [32]. Both methods present, however, a major drawback: they require contact with the biological material, making measurements time and labour consuming when large numbers of plants must be evaluated or when the plants are in difficult to access areas. PEA also requires a darkness adaptation period for measuring the minimal fluorescence intensity, making the tests slow.

One of the most important problems in Mediterranean forest management is the exact evaluation of the impact of forest fires and extended drought periods on plants health. The accurate assessment ofwater scarcity related stress is also of utmost importance for irrigation optimization, particularly taking into consideration the increasing global water deficit [23]. A fast, remote and contactless method of plant stress evaluation would greatly facilitate the effective inspection of large woods and plantations. The aim of the present work was to develop a method for the remote assessment of plants drought and fire stress based on laser-induced fluorescence analysis (LIF-LIDAR). The method was applied to two species of paramount importance in Mediterranean forestry, namely cork oak (Quercus suber) and maritime pine (Pinus pinaster).

The proposed method was benchmarked against PEA and water loss measurements.


The experiments were carried out using the LIF-LIDAR setup represented schematically in Fig. 1. Fluorescence is excited by the second harmonic of a pulsed YAG:Nd laser (X = 532 nm) with a pulse duration of 5 ns, maximum energy per pulse 20 mJ and pulse repetition rate 10 Hz. The fluorescent radiation is collected by a 20 mm diameter telescope. To prevent backscattered laser radiation from entering the telescope and saturate or damage the spectrometer CCD, a long pass filter with a cut-off wavelength of 550 nm was introduced in the optical path, in front of the telescope. The collected radiation is transferred to a low-noise computer-controlled CCD spectrometer (Ocean Optics USB4000) by an optical fibre. In order to reduce the influence of background radiation on the measurements, the spectrometer was operated in the external triggered mode, using short time intervals (about 10 ^s) synchronized with the laser pulse emission for data acquisition. To increase the signal-to-noise ratio, each spectrum results from averaging radiation collected during 10 laser pulses. The distance between the LIF-LIDAR instrument and the samples was 1 m.

Analysis of the accuracy of the fluorescence intensity measurements indicated that the main sources of errors are the instability of the laser-pulse energy and the nonuniformity of the CCD-detector response. For the equipment involved in the experiments, the total relative error of the measurements did not exceed 10% for the LIF intensities and 7% for the integral-fluores-cenct parameter Fv /Fm.

Normalized, signal 1.0r


s2 days

Start of the experiments

Fv /Fm 1.0




800 X, nm

Fig. 2. Fluorescence spectra of cork oak leaves.


Ten leaves of mature cork oak and 40 maritime pine needles were used for each series of experiments. The leaves were collected immediately before the experiments and stored at 22°C and 50% humidity until complete drying. Maritime pine needles required about 11 days for complete drying, while the cork oak leaves loose water much faster, in about 2.5 days. During the drying period the leaves were periodically removed from the controlled humidity oven and submitted to the following tests: weight measurement using an analytical balance with a precision of 0.1 mg; evaluation of F0 and Fv/Fm ratio using a Handy PEA fluo-rometer; fluorescence spectral analysis using the instrument represented in Fig. 1. The measurements were performed once a day on the maritime pine needles and twice a day on the oak tree leaves. The first experiments showed that pine tree needles do not loose water uniformly, water loss being slower near the needle sheath than near its tip. To take this into consideration, measurements were performed in two different positions along the needle length: near the sheath, and at mid-length. In general the values presented are the average of 10 measurements. The water loss was characterized by the relative water content (RWC), given by [33]:

F0, a.u 600

400 -

200 -




I685/I740 1.0


Time, days

Fig. 3. Fv/Fv (a), F0 (b), and I685/I740 (c) vs time for cork oak leaves. T

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