Dualex: a new instrument for field
measurements of epidermal ultraviolet
absorbance by chlorophyll fluorescence
Yves Goulas, Zoran G. Cerovic, Aurélie Cartelat, and Ismaël Moya
Dualex 共dual excitation兲 is a field-portable instrument, hereby described, for the assessment of polyphenolic compounds in leaves from the measurement of UV absorbance of the leaf epidermis by double
excitation of chlorophyll fluorescence. The instrument takes advantage of a feedback loop that equalizes
the fluorescence level induced by a reference red light to the UV-light-induced fluorescence level. This
allows quick measurement from attached leaves even under field conditions. The use of light-emitting
diodes and of a leaf-clip configuration makes Dualex a user-friendly instrument with potential applications in ecophysiological research, light climate analysis, agriculture, forestry, horticulture, pest management, selection of medicinal plants, and wherever accumulation of leaf polyphenolics is involved in
plant responses to the environment. © 2004 Optical Society of America
OCIS codes: 120.6200, 120.7000, 170.6280, 170.6510, 300.6540, 230.3670.
1. Introduction
Higher-plant cells contain not only basic life molecules such as carbohydrates, proteins, lipids, or nucleic acids but also a wide range of other molecules
with a phenolic chemical structure often called plant
polyphenolics 共Phen兲. The number of these compounds identified to date may now exceed 100,0001;
some of them occur in only one or a few species.
Plant Phen first appeared to have no intrinsic role in
the physiological process and were classified as secondary metabolites. But over the past 40 years, as
the knowledge about their chemical structure and
their biochemical interactions increased, the interest
in the biological role of these compounds increased,
together with the identification of specialized enzymes responsible for their synthesis under strict genetic control. Some of their roles in plants were
found to be related to the protection against harmful
UV radiation,2,3 plant– herbivore interactions,4 or a
The authors are with the Equipe Photosynthèse et Télédétection, Laboratoire pour l’Utilisation du Rayonnement Electromagnétique, Centre National de la Recherche Scientifique, Batiment
203, Centre Universitaire Paris-Sud, B.P. 34, F91898 Orsay Cedex, France. The e-mail address of Y. Goulas is
[email protected].
Received 17 December 2003; revised manuscript received 10
May 2004; accepted 17 May 2004.
0003-6935兾04兾234488-09$15.00兾0
© 2004 Optical Society of America
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APPLIED OPTICS 兾 Vol. 43, No. 23 兾 10 August 2004
chemical defense against pests and pathogens.5
Some attention was also given to the possible use of
Phen in agriculture, forestry, food industry, and medicine. In this context, the development of analytical
instruments aimed at the assessment of polyphenolics in plants is of particular importance.
Phen compounds have typical UV absorption peaks
in the UV-A and UV-B 共for a compilation, see Ref. 6兲.
Because of the large number of compounds absorbing
in the UV, a low reflectance, and an almost zero
transmittance of leaves in this spectral region, the
classical spectroscopic method cannot be easily applied.7 As a large proportion of leaf Phen has been
shown to be present in the epidermis, alternative
spectroscopic methods have emerged for in situ assessment of phenolics in leaves. In this paper the
term epidermis is used in its wider sense, encompassing the cuticle, the proper epidermis, and the hypodermis. For instance, UV absorbance of epidermal
peels was shown to be correlated to the total content
of UV-absorbing compounds in leaves.8 Another approach was proposed that uses a thin optical fiber
penetrating gradually into the leaf to measure UV
transmittance of the epidermis and the mesophyll.9
More recently Bilger et al.10 proposed the use of
UV-induced chlorophyll fluorescence 共ChlF兲 to assess
the UV-absorbing properties of the epidermis with a
Xe-PAM fluorometer 共Walz, Effeltrich, Germany兲 and
later11 with a specific instrument, the UV-A-PAM
共Gademann Instruments, Würzburg, Germany兲.
The ratio of UV-induced ChlF to blue-green 共BG兲induced ChlF 关F共UV兲兾F共BG兲兴 was found to correlate
with the epidermis Phen content in several species
共see also Ref. 12兲.
The main assumption of the method is that ChlF
yield remains constant between UV- and BG-induced
ChlF measurements. However, as it is well known,
ChlF varies as a consequence of changes in photochemical and nonphotochemical quenching.13 In
the Xe-PAM approach,10 one can prevent variations
of ChlF yield, first, by keeping the measured leaves in
the dark for at least 30 min before the measurements
to reach a stable dark level of ChlF and, second, by
using a low level of measuring light to avoid any ChlF
induction by the measuring beam 共the Kautsky effect,
cf. Ref. 13兲. This method requires a long measuring
time and laboratory conditions. The UV-A-PAM approach uses rapidly alternating UV-A and blue LED
emissions, such that F共UV兲 and F共BG兲 are monitored
quasi-simultaneously. But again, a low-intensity
measuring beam is used in this device to avoid affecting ChlF. So, larger leaf Phen contents, and therefore larger UV screening, will need either a stronger
measuring light or a longer measuring time to attain
the same level of accuracy.
Another drawback of the above-described devices is
the use of a blue 共in UV-A-PAM兲 or BG 共in Xe-PAM兲
light as the reference beam. Indeed, Barnes et al.14
noticed in some species an increase in the F共UV兲兾
F共BG兲 ratio on UV-B treatment, instead of the commonly observed decrease of this ratio, as a
consequence of UV-absorbing compounds accumulating in the epidermis. This anomalous variation can
be the consequence of the accumulation of absorbing
compounds even in the BG spectral region.
In this paper we present the design and utilization
of a new instrument, the Dualex 共dual excitation兲, for
the measurement of epidermal UV absorption of
leaves by ChlF. The Dualex uses an original feedback loop to fully eliminate any effects of variable
ChlF. Thus the intensity of the measuring light is
no longer limited by its effects on variable ChlF, so a
wider range of epidermis absorbances can be assessed. In addition, a red reference light has been
chosen for which the epidermis is almost always
transparent. The Dualex can be used for rapid 共less
than a second兲 outdoors or laboratory measurements.
The chosen geometry with back-face detection avoids
the use of light guides that makes the Dualex a robust and low-cost device.
2. Measurement Principle
Measurement of leaf epidermal transmittance of UV
light by ChlF is based on the screening effect of the
epidermis to incident UV light, therefore decreasing
the amount of light available for ChlF excitation.
Most of this absorption is caused by strong UV absorbers, mainly localized in leaf epidermis.3,10 The
epidermal transmittance is assessed by the fluorescence excitation ratio F共UV兲兾F共REF兲, where F共UV兲 is
the fluorescence detected following UV excitation and
F共REF兲 is the fluorescence detected on excitation at a
Fig. 1. Emission spectra of the UV and the reference red-light
sources compared with absorption spectra of chlorophylls and fluorescence spectra of leaves. 共a兲 Absorption spectra of chlorophyll
a 共Chl a兲 and chlorophyll b 共Chl b兲 in methanol 共MeOH兲. 共b兲
Fluorescence excitation spectrum of a wheat leaf 共emission wavelength 740 nm, total Chl content 50 ␮g兾cm2兲. 共c兲 Emission spectra
of filtered UV and red-light sources compared with the back-side
leaf ’s Chl fluorescence emission 共excitation wavelength 375 nm兲
and the transmittance spectrum of the emission filter 共dashed
curve兲.
reference wavelength, not absorbed by the epidermis.
In this setup, chlorophyll in the mesophyll acts as a
photosensor providing a fluorescence emission in response to the incident light reaching the mesophyll
共see Fig. 1 for absorption, excitation, and emission
spectra兲. The method is based on the assumption of
10 August 2004 兾 Vol. 43, No. 23 兾 APPLIED OPTICS
4489
a constant ratio among different samples between the
intrinsic ChlF yields excited by UV and the reference
wavelength.
Given
• I共UV兲 and I共REF兲, the excitation light intensities at the leaf level in the UV and at the reference
wavelength, respectively,
• ␾共UV兲 and ␾共REF兲, the apparent ChlF yields of
the mesophyll excited by the UV and the reference
wavelengths, respectively,
• T共UV兲, the epidermal transmittance in the UV,
• an epidermal transmittance at the reference
wavelength assumed to be 1,
we can then write
F共UV兲
␾共UV兲 I共UV兲
⫽ T共UV兲
.
F共REF兲
␾共REF兲 I共REF兲
(1)
Three problems have to be solved to obtain T共UV兲
from the F共UV兲兾F共REF兲: 共1兲 the presence of variable ChlF, 共2兲 the variability in leaf Chl content in
different samples, and 共3兲 the changes in ChlF yield of
photosystem I 共PSI兲 and photosystem II 共PSII兲.
A.
Variable Chlorophyll Fluorescence
Epidermis absorbance measurement by ChlF is
based on the comparison between two ChlF levels.
Therefore any unwanted variation during the measurements of these two ChlF levels due to changes in
ChlF yield would compromise the result.
With Dualex, we are able to fully eliminate any
artifact caused by variable ChlF by the following
method:
• Leaves are illuminated by two rapidly alternating light sources at a frequency of 1 kHz;
• The light intensity of the UV source is maintained at a fixed level, while the light intensity of the
reference red source is constantly adjusted by a feedback loop so that the UV-induced ChlF and the
reference-light-induced ChlF remain equal.
In this way, the fluorescence excitation ratio F共UV兲兾
F共REF兲 is equal to 1, and the light intensity of the
reference source I共REF兲 can be used to deduce the
epidermal transmittance, provided that the ␾共UV兲兾
␾共REF兲 ratio is constant. Equation 共1兲 becomes
T共UV兲
␾共UV兲
I共UV兲 ⫽ I共REF兲.
␾共REF兲
(2)
Let us call I0共REF兲 the excitation light intensity at
the reference red wavelength measured in a standard
experimental situation in which the transmittance
T共UV兲 is conventionally taken as 1. Such an actual
standard situation will be described later. Then, by
writing Eq. 共2兲 for the measurement and standard
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APPLIED OPTICS 兾 Vol. 43, No. 23 兾 10 August 2004
situation and dividing the two equations member to
member, we can write
T共UV兲 ⫽
I共REF兲
.
I 0共REF兲
(3)
The epidermal UV absorbance A is defined as the
base-10 logarithm of the transmittance reciprocal:
A ⫽ ⫺log10
冋
册
I共REF兲
.
I 0共REF兲
(4)
So, epidermal UV absorbance is measured directly
from the light intensity of the reference source. This
method, in addition to the elimination of variable
ChlF effects, has the advantage of providing, after
proper calibration, a single analogous signal representative of epidermal UV transmittance. In the
portable version of the apparatus, the output signal is
simply converted into absorbance with an analog logarithmic converter and sent to a digital display. Details on the functioning of the circuits are given later.
B.
Leaf Chlorophyll Content
We are looking for situations in which the ChlF yield
ratio ␾共UV兲兾␾共REF兲 does not depend on leaf chlorophyll content. This will be the case if we choose two
wavelengths with equal molar-extinction coefficients.
This will also be the case in leaves with high Chl
content, for strongly absorbed wavelengths, and if the
molar-extinction coefficients remain close to each
other. One can see that the peaks’ amplitude is
strongly attenuated in the ChlF excitation spectrum
关Fig. 1共b兲兴 where Chl content is high, compared with
the absorption spectra of a dilute solution 关Fig. 1共a兲兴.
Indeed, Bilger et al.10 showed that the ratio F共UV兲兾
F共BG兲 under UV-A excitation does not depend on leaf
Chl content.
C. Chlorophyll Fluorescence Yields of Photosystems I
and II
Chlorophyll fluorescence originates from both PSI
and PSII. PSII fluorescence is variable, whereas
that of PSI is not13 or very little.15 PSI and PSII also
have different ChlF excitation and emission spectra,
owing to a difference in their chlorophyll a兾chlorophyll b 共Chl a兾Chl b兲 ratio. In our instrument, a
high-pass far-red filter is used to separate fluorescence emission from the excitation light 关see Fig.
1共c兲兴, so the detected ChlF has contributions from
both photosystems.13
On the one hand, if the relative proportions of PSI
and PSII emissions in the detected ChlF are wavelength dependent, variations of PSII quantum yield
during kinetic induction will result in variations of
the ␾共UV兲兾␾共REF兲 ratio and will affect the measurements. On the other hand, a constant ␾共UV兲兾
␾共REF兲 ratio will be maintained if we use excitation
wavelengths at isosbestic points 共equal molarextinction coefficients兲 for Chl-a and Chl-b absorption. Moreover, as Chl b is a minor absorber, it
would be sufficient to avoid the 450 – 490-nm region,
characterized by a strong absorption of Chl b and a
weak absorption of Chl a. This strategy will also
prevent other effects linked to PSI–PSII heterogeneity, such as the variations of Chl-a and Chl-b distribution between PSI and PSII, seen among samples or
induced on a single leaf by the state I–state II transition.13
D.
Choice of Excitation Wavelengths
Following the above considerations, it can be seen
from Fig. 1共a兲 that the best choice of wavelength is
around 350 nm for the UV light and 650 nm for the
reference. Chl-a and Chl-b absorptions are nearly
equal at these wavelengths. One should keep in
mind that absorption spectra in Fig. 1共a兲 are that of
Chl in MeOH—a rough approximation of the true
absorption spectra in vivo. Technological and physiological constraints must also be taken into account.
UV LEDs, which became commercially available recently, offer the opportunity to build a small and
simple instrument. These light sources are easily
modulated and simplify the electronic design. Several models of the UV LED are available with emission wavelengths at 350, 370, and 380 nm. The
chosen LED, UVLED370-10 共Roithner Lasertechnik,
Vienna, Austria兲, is a compromise between wavelength 共375 nm, 15-nm FWHM, measured values兲
and optical power 共0.75 mW兲. The main drawback
at this wavelength is the absorption ratio for Chl a
and Chl b, which is 80% higher for Chl a than Chl b.
On the other hand, because of the large content of
UV-absorbing compounds in leaves exposed to the
full sun, it is better to use a longer-wavelength UV,
which will yield a stronger ChlF signal. The reference wavelength must be set to an equally absorbed
wavelength in the red. This condition can be met by
red LEDs at 657 nm, such as the URC383-3 共Roithner
Lasertechnik, Vienna, Austria兲, or at 655 nm, such as
the MV8114 共Fairchild Semiconductor, South Portland, Maine, USA兲.
3. Description of the Dualex
The instrument16 is built in a modular way. The
three main parts are an electro-optical head forming
a clip enclosing the leaf 共Fig. 2兲; an electronic module
for signal decoding, LEDs driving, and peripheral
interfacing; and an interface module comprising a
digital display and a battery power supply.
A portable data logger can also be connected to the
interface, for data storing and subsequent transfer to
a microcomputer. The electro-optical clip comprises
all optical parts, light sources, filters, the detector,
and the detector preamplifier. It is connected
through an electric cable to the electronic module.
The whole system is portable and adapted to measurements in field conditions.
In its laboratory configuration, an acquisition and a
timer card 共CIO-DAS802兾16 and CIO-CTR05, respectively, Computers Boards, Mansfield, Maine, USA兲
are installed on a personal-computer-type microcomputer and connected to an electronic module. In this
mode of operation, automated data acquisition and
Fig. 2. Utilization of the Dualex clip for measurement of the
epidermis leaf ’s UV absorbance.
easy visualization can be obtained from a proprietary
software developed in VEE language 共VEE 5.0, Agilent
Technologies, Palo Alto, California, USA兲 that transmits acquired data to a plotting and storing environment 共Igor, Wavemetrics, Lake Oswego, Oregon,
USA兲.
A schematic drawing of the apparatus, in the
microcomputer-controlled mode of operation, is presented in Fig. 3. It comprises the UV LED and the
reference red LED. The UV light is filtered with a
2-mm-thick DUG11 filter 共Schott, Clichy, France兲,
and both lights are filtered with a short-pass interference filter 共custom made, Omega, Brattleboro,
Vermont, USA兲 placed between the LED sources and
the leaf. Figure 1共c兲 shows the diodes’ emission
spectra after filtering at the leaf level. The two diodes are tilted to illuminate a common area on the
leaf of approximately 5 mm in diameter. Far-red
ChlF from the leaf is detected from the other side of
the leaf by a silicon photodiode, which is followed by
a transimpedance amplifier and a high-pass filter.
An UV-blocking filter 共KV420, Schott, Clichy, France兲
and a far-red long-pass filter 共RGN9, 4 mm thick,
Schott, Clichy, France兲 are placed between the leaf
and the detector to block any excitation light and to
ensure detection of far-red ChlF only. The advantage of this design is that no additional collecting
optics are required.
A.
Functioning of the Instrument
The two diodes emit light pulses at a frequency of 1
kHz, and the duration of the pulses is half of the
period. Each light source is phase shifted at 180°
from the other, so that the leaf is alternatively illuminated by UV and red light at the modulation frequency 关Fig. 4共a兲兴. The detected fluorescence signal
10 August 2004 兾 Vol. 43, No. 23 兾 APPLIED OPTICS
4491
Fig. 3. Schematic diagram of the Dualex. A feedback loop minimizes the difference between UV-induced and red-induced fluorescence, canceling effects caused by variable chlorophyll
fluorescence. AD, analog-to-digital.
is preamplified and demultiplexed by two sampleand-hold circuits 共Fig. 3兲. Each sample-and-hold
circuit is synchronized so that a signal corresponding
to the amplitude of a UV-induced fluorescence level is
present at one input of a differential amplifier and a
signal corresponding to the amplitude of a redinduced fluorescence level is present at the other input. The difference between these two signals is
then sent to a high-gain low-pass amplifier 共cut frequency f3dB ⫽ 40 Hz兲, with the output delivered to the
red-LED driver. The LED current is proportional to
the control voltage at the input of the LED driver.
Thus if the UV-induced ChlF level becomes smaller
than the red-induced fluorescence level, the red-LED
control voltage will decrease, and the difference between the two fluorescence levels will decrease. The
described feedback loop constantly minimizes the difference between the UV-induced fluorescence F共UV兲
and the red-induced fluorescence F共R兲. Because of
the high gain of the feedback loop 共⬎106兲, the difference remains far below the noise level and can be
neglected. In addition, the amplitude of the UV
pulses, I共UV兲, remains constant. Hence switching
from UV light to red light, and vice versa, does not
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APPLIED OPTICS 兾 Vol. 43, No. 23 兾 10 August 2004
Fig. 4. Induction kinetics of chlorophyll fluorescence emission as
seen by the detection photodiode 共solid curve兲 and of the mean
red-light level 共dashed curve兲. 共a兲 Illustration of the feedback loop
functioning 共short time scale兲. At time t ⫽ 0, UV light is suddenly
increased. The fluorescence level then oscillates between the UVinduced level 共0.5-ms pulses, top of the curve兲 and the red-induced
level 共0.5-ms inversed pulses, bottom of the curve兲 until the two
levels become equal after 15 ms. 共b兲 Measurement on a leaf. No
oscillations of fluorescence emission can be seen. After 300 ms of
illumination, the power of the red LED, which is a measure of
epidermal UV absorbance, remains constant despite a large variation in chlorophyll fluorescence.
induce any changes in the ChlF level, and the observed slow changes in ChlF 关Fig. 4共a兲兴 are due to the
already mentioned ChlF induction 共Kautsky effect兲.
They do not interfere with the measurement of UV
absorbance because the variations are much slower
than the modulation period of the LEDs 关Figs. 4共a兲
and 4共b兲兴. We find that, during one period of modulation, the ChlF yield during red excitation is equal to
the ChlF yield during UV excitation, and so the
␾共UV兲兾␾共R兲 ratio remains constant 关here ␾共R兲 ⫽
␾共REF兲兴. Therefore Eq. 共4兲 holds, and the measurement of red-light intensity is sufficient to assess the
epidermal UV absorbance.
Fig. 5. Optical power of the red LED as a function of the driving
current 共log–log scale兲. The relationship is not linear over several
decades.
B.
Measurement of Red-Light Intensity
Light intensity of the red reference LED could be
obtained from the driving current. Unfortunately,
the light intensity emitted by the LED is not proportional to the driving current over a large range of
current variation. It presents an almost linear relationship in the milliampere range but an almost
quadratic one in the 10 –100-␮A range 共Fig. 5兲. One
method to assess the light intensity would be to digitize the control voltage of the red-LED driver and to
use a previously recorded calibration curve of the
radiated power as a function of the driving current.
This solution would need a computing device absent
from our instrument. Instead, we chose to measure
the small amount of internally reflected light from
the red reference LED with a small photodiode fixed
on its back. The resulting amplified signal is transformed into a continuous signal representative of the
mean light intensity of the red LED. In the computer mode of operation, the latter signal is digitized
with an analog-to-digital conversion card, and in the
portable version of the instrument, it is converted
with a logarithmic amplifier.
C.
Feedback Loop
Figure 4共a兲 shows the kinetics of ChlF changes of a
fluorescence standard 共Walz standard, Walz, Effeltrich, Germany兲 after a rapid change in UV-light intensity. This is an illustration of the feedback loop
functioning. Before time t ⫽ 0, UV- and red-induced
ChlF levels are equal as a result of the feedback loop
action. At time t ⫽ 0, the UV-light intensity is suddenly changed. The feedback circuit becomes unbalanced, and UV-induced ChlF is larger than redinduced ChlF. This results in a modulation of the
fluorescence signal at the same frequency as the light
sources. Within 15 ms, the feedback circuit in-
creases the current in the red diode until UV- and
red-induced ChlF become equal. Figure 4共a兲 shows
the corresponding increase of red-light intensity
共right-hand scale兲.
Figure 4共b兲 shows the kinetics of ChlF and of redLED light intensity on illumination of a dark-adapted
leaf by the combination of UV light and the feedbackcontrolled red light. Except at the very start of the
curve, the fast 1-kHz modulation of the ChlF signal is
not detectable, even in the presence of variable ChlF.
The increase in ChlF 关Fig. 4共b兲兴 reflects the photochemical reduction of the PSII acceptor QA and is
similar to the one that would be obtained under illumination by continuous light. This variable ChlF is
slow as compared with the modulation frequency of
the UV and red lights. As a result, the corresponding variations of the ChlF yield within a period of
modulation of UV and red lights can be neglected. It
can be seen in Fig. 4共b兲 that the red-LED light intensity is constant during the ChlF induction kinetics,
except for the first 200 ms showing a slight variation
that will be discussed later. Hence variations in
ChlF yield affects identically the UV- and the redlight-induced ChlF. In the computerized version,
I共REF兲 is sampled several times during its stationary
phase 共approximately 200 times兲, and the mean value
of the samples is calculated to optimize measurements’ repeatability. The illumination time is approximately 0.6 s per measurement. In the portable
version, light diodes are continuously switched on.
An analog logarithmic converter is used, and the true
value of absorbance is continuously displayed. It
should be mentioned here that the dark-to-light transition depicted in Fig. 4 is an extreme case that does
not occur in normal use of the portable instrument in
the field.
In Fig. 4共b兲, a small variation of I共REF兲 can be seen
during the first 200 ms. This variation disappears
when the leaf is replaced by the fluorescence standard 共the Walz plastic foil兲. Therefore it is not
caused by an instrumental unbalancing between the
UV and the red channels, such as, for example, a
variation of diode emission yield at the beginning of
the illumination period. Neither can it be attributed
to the activation of the feedback loop because it disappears when the UV LED is replaced by a red LED
identical to the red reference LED, even though the
variable ChlF remains under these conditions. Furthermore, the association of different couples of wavelength in the UV, blue, yellow, green, and red results
in different shapes of this transient 共data not shown兲.
So, this variation in red-LED intensity, associated
with the so-called I phase of the ChlF induction kinetics 共cf. Ref. 13兲, must be interpreted as a variation
in the relative efficiency of ChlF excitation by the UV
and the red lights. Several known phenomena can
be involved in this wavelength-dependent excitation
induction: pigment–protein complexes’ transfer between the two photosystems, induction of quenchers
caused by a change in the redox state or in the pH of
the thylakoid lumen, and an electrochromic effect 共cf.
Ref. 17兲. Further studies of this wavelength10 August 2004 兾 Vol. 43, No. 23 兾 APPLIED OPTICS
4493
path cell. This concentration corresponds to a surface content of approximately 27 ␮g兾cm2 Chl a, which
is an average content found in leaves.
B.
Calibration by Use of Filters of Known Absorbance
To test the accuracy of Dualex measurements, we
simulated an increase in leaf epidermis UV absorbance by covering the leaf with filters of known increasing UV absorbances. The filters used were
thin colored plastic foils from Lee Filters 共Andover,
England兲. Using different combinations from six filters 共reference numbers 147, 148, and 151–154兲, we
obtained a UV absorbance ranging from 0.17 to 2.2.
The small absorbance of the filters in the red region
of the spectrum, measured with a diode-array spectrophotometer 共HP8453, Hewlett-Packard, USA兲,
was corrected for. A linear relationship up to an
absorbance of 2.2 was obtained between the Dualex
and the spectrophotometer values 共Fig. 6兲.
Fig. 6. Absorbance measurement of different combinations of
thin plastic filters. We obtained Dualex values by covering an
indoor-grown leaf with the filters 共uncovered leaf value ⫽ 0.03兲.
Spectrophotometer values were measured at 30° incidence. Error
bars are standard deviations of the mean 共visible only on the last
point兲.
C.
Sources of Variability
dependent variation of the ChlF excitation efficiency
would be interesting, but they are beyond the scope of
this paper.
Table 1 presents an estimation of variability that can
affect Dualex measurements. Small changes in the
optical head position are a source of error eight times
larger than short-term instrumental variations. Table 1 also shows that Dualex values can vary up to a
factor of 2 along the leaf surface. This fact must be
taken into consideration when one sets up methodological procedures with the Dualex instrument.
4. Measurements and Applications
D.
A.
Calibration Procedure
The constant I0共REF兲 in Eq. 共4兲 can be determined by
one’s considering a reference situation in which the
UV absorbance is known. Absolute values of epidermis UV absorbance are difficult to obtain by absorption spectrometry. Leaves with the epidermis
removed still have UV-absorbing compounds in the
mesophyll at different concentrations depending on
the leaf sample. A more stable and reliable reference is obtained with a solution of Chl a in MeOH, for
which there is no UV screening at all. A high
enough Chl concentration was chosen that will absorb all excitation light in the UV and in the red.18
We choose a 150-␮M solution in a thin 2-mm optical
Examples of Application
The use of instruments designed to measure epidermal UV absorbance in order to assess the degree of
UV protection of leaves is well documented 共cf. Ref.
11兲, but new applications have emerged for this type
of instrument. Because a large portion of Phen responsible for UV absorbance is present in the epidermis 共see Section 1兲, Dualex measurements can be used
to estimate the Phen content of leaves. For example, we compared the Phen content on leaf extracts
共30 min in MeOH, 70 °C兲 to Dualex values obtained
from the same wheat leaves grown with different
nitrogen fertilization rates 共see also Ref. 19兲. Dualex values were obtained by summing measurements
from both adaxial and abaxial sides of the leaf. The
results in Fig. 7 show that a good correlation exists
Table 1. Sources of Measurements’ Variability
Source of Variation
Instrumental variationsa
Position repeatabilityb
Leaf surface heterogeneityc
共Morus nigra L.兲
共Triticum aestivum L.兲
Mean
共absorbance units兲
Minimum
Maximum
Standard Deviation
共absorbance units兲
Relative Standard
Deviation
0.289
0.287
0.288
0.284
0.290
0.291
0.0003
0.0024
0.1%
0.8%
0.357
2.06
0.258
1.93
0.491
2.24
0.06
0.10
17%
5%
a
Short-term 共⬍1 h兲 instrumental variations: successive measurements on the same point without movement of the optical head 共Acer
pseudoplatanus兲.
b
Positioning repeatability: measurements on the same point with removal and replacing of the optical head between measurements
共Acer pseudoplatanus兲.
c
Surface leaf heterogeneity: measurements on different points of the same leaf.
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APPLIED OPTICS 兾 Vol. 43, No. 23 兾 10 August 2004
received by a leaf as exemplified by the results presented here 共Fig. 8兲 for a beech tree 共Fagus silvatica
L.兲.
5. Discussion
The use of fluorescence to measure absorbance of
opaque samples in the UV can be traced to the work
of Amesz and co-workers22 in the early sixties, but it
was fully applied to leaves by Bilger et al.10 Recently the same authors have described a portable
device, which they call UV-A-PAM,11 that was designed to acquire the same type of information as the
Dualex. Still, the major differences between the two
instruments are as follows:
Fig. 7. Comparison of Dualex values with wheat leaf extracts
共absorbance at 375 nm兲. Dualex values are the sum of adaxial
and abaxial side measurements. Absorption spectra were corrected in the UV for Chl-a and Chl-b contributions.
between Dualex-derived UV absorbance and the extract absorbance. The intercept on the Dualex axis
共at zero extract absorbance兲 can be explained by the
presence of UV-absorbing compounds in the cell walls
not extractable by hot methanol.20
From the strong correlation between Phen assessed with the Dualex and the leaf mass per area
共Fig. 8兲, one can conclude that Phen could also be
used as a nondestructive indicator of light distribution in the canopy. Indeed, the leaf mass per area is
known to be a good indicator of irradiance received by
the leaves during their development in a tree canopy.21 Phen could also be a good indicator of the light
Fig. 8. Phen in leaves of a beech tree. Correlation between the
total Phen content of leaves measured with the Dualex and their
leaf mass per area.
• The use of a blue reference light in UV-A-PAM,
compared with a red one in Dualex;
• Front-face measurements by use of a light
guide, compared with back-side measurements;
• The use of low measuring light intensity, compared with a much stronger light in Dualex.
The UV-A-PAM device has been proposed mainly as
a tool to assess the protection from UV radiation in
ecophysiological research, but both the Dualex and
the UV-A-PAM have wider potential applications.
Indeed, because of their preferential presence in
the epidermis and their spectral characteristics, the
flavonoids are the major compounds contributing to
absorption measured by the Dualex in the UV-A region. Therefore many of the known functions of flavonoids in the leaf could be probed with the Dualex:
protection from UV irradiation; defense against
pathogen attacks; defense against herbivores,
whether insect or vertebrate; protection from oxidative stress 共free-radical scavenging兲; metal chelation;
and drought resistance. But some other plant functions or physiological states that influence the flavonoid content in the leaf, such as low temperature,
the onset of senescence, or low availability of nitrogen
in the soil, could also be assessed by the Dualex.
Indeed, both the carbon–nutrient balance4 and the
growth– differentiation balance23 hypotheses predict
an increased synthesis of carbon-based quantitative
secondary compounds 共mainly Phen兲 in the leaf in
nutrient-limited environments. So, because Phen,
in general, and flavonoids, in particular, contribute to
the UV absorption of the leaf, their content can be
assessed with the Dualex and used as a proxy for leaf
nitrogen.19,24 This new indicator, nondestructively
accessible with the Dualex, is complementary to the
nitrogen diagnosis of crops based on chlorophyll measurements with the N-Tester 共Hydro-Agri兲 or SPAD
共Minolta兲 devices. The combined information
gained from both chlorophyll and Phen leaf content
will help in the design of new and better decisionsupport systems for agriculture.
Measurements of Phen with the Dualex can also be
used to characterize the light climate in a canopy, for
example, in forestry research. Indeed, there is a
strong positive relationship between the accumulation of Phen and light intensity, without known ex10 August 2004 兾 Vol. 43, No. 23 兾 APPLIED OPTICS
4495
ceptions.1 This is usually explained by the abovementioned carbon–nutrient balance hypothesis
because light is acting through photosynthesis as a
carbon-rich source. In summary, in addition to ecophysiological research, the Dualex can find applications in agriculture, forestry, and horticulture, for
nitrogen assessment and fertilization, pest defense
management, light climate analysis, selection of medicinal plants, and wherever leaf Phen are directly or
indirectly involved in plant responses.
Further developments of the Dualex will mainly be
related to the availability of new UV-emitting LEDs,
in order to extend the measurements to the
hydroxycinnamates-absorbing band 共320 nm兲 or even
to shorter wavelengths, although it will be more difficult to attain the required sensitivity owing to stronger absorption. The possibility for Dualex to use
strong light, thanks to its feedback loop, will certainly
be an asset in this case. Otherwise, the absorbers
present in the visible part of the spectrum, such as
anthocyanins, can also be probed through our
method, thanks to the availability of green LEDs, but
in this spectral region the low absorbance of the chlorophyll might be a problem that should be taken into
account by adequate modeling.
The authors thank M. Bergher for reviews and
suggestions on electronic circuits, P. Minola and J. F.
Vagnucci for electronic circuit routing and cabling, J.
Walocha for mechanics realization, and N. Tremblay
for a critical reading of the manuscript. Measurements relative to leaf mass area were conducted in
cooperation with E. Dreyer from the Institut National
de la Recherche Agronomique, Nancy, France. Dualex was designed and made possible by the financial
support of the Centre National de la Recherche Scientifique through Groupement de Recherches 1536
FLUOVEG 共fluoresence vegetation兲 dedicated to the
measurement of vegetation productivity by fluorescence.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
References
1. P. G. Waterman and S. Mole, Analysis of Phenolic Plant Metabolites, Methods in Ecology 共Blackwell Scientific, Oxford,
UK, 1994兲, p. 238.
2. M. M. Caldwell, “Solar UV irradiation and the growth and
development of higher plants,” in Photophysiology, A. C. Giese,
ed. 共Academic, New York, 1971兲, pp. 131–171.
3. M. M. Caldwell, R. Robberecht, and S. D. Flint, “Internal filters: prospects for UV-acclimation in higher plants,” Physiol.
Plant. 58, 445– 450 共1983兲.
4. P. D. Coley, J. P. Bryant, and S. Chapin, “Resource availability
and plant antiherbivore defense,” Science 230, 895– 899
共1985兲.
5. K. Hahlbrock and D. Scheel, “Physiology and molecular biology
of phenylpropanoid metabolism,” Annu. Rev. Plant Physiol.
Plant Mol. Biol. 40, 347–369 共1989兲.
6. Z. G. Cerovic, A. Ounis, A. Cartelat, G. Latouche, Y. Goulas, S.
Meyer, and I. Moya, “The use of chlorophyll fluorescence excitation spectra for the non-destructive in situ assessment of
UV-absorbing compounds in leaves,” Plant Cell Environ. 25,
1663–1676 共2002兲.
7. M. N. Merzlyak, A. E. Solovchenko, and A. A. Gitelson, “Reflectance spectral features and non-destructive estimation of
4496
APPLIED OPTICS 兾 Vol. 43, No. 23 兾 10 August 2004
20.
21.
22.
23.
24.
chlorophyll, carotenoid and anthocyanin content in apple
fruit,” Postharvest Biol. Technol. 27, 197–211 共2003兲.
T. A. Day, B. W. Howells, and W. J. Rice, “Ultraviolet absorption and epidermal-transmittance spectra in foliage,” Physiol.
Plant. 92, 207–218 共1994兲.
J. F. Bornman and T. C. Vogelmann, “Penetration of blue and
UV radiation measured by fiber optics in spruce and fir needles,” Physiol. Plant. 72, 699 –705 共1988兲.
W. Bilger, M. Veit, L. Schreiber, and U. Schreiber, “Measurement of leaf epidermal transmittance of UV radiation by chlorophyll fluorescence,” Physiol Plant 101, 754 –763 共1997兲.
W. Bilger, T. Johnsen, and U. Schreiber, “UV-excited chlorophyll fluorescence as a tool for the assessment of UV-protection
by the epidermis of plants,” J. Exp. Bot. 52, 2007–2017 共2001兲.
P. Burchard, W. Bilger, and G. Weissenbock, “Contribution of
hydroxycinnamates and flavonoids to epidermal shielding of
UV-A and UV-B radiation in developing rye primary leaves as
assessed by ultraviolet-induced chlorophyll fluorescence measurements,” Plant Cell Environ. 23, 1373–1380 共2000兲.
G. Krause and E. Weis, “Chlorophyll fluorescence and photosynthesis: the basics,” Annu. Rev. Plant Physiol. Plant Mol.
Biol. 42, 313–349 共1991兲.
P. W. Barnes, P. S. Searles, C. L. Ballare, R. J. Ryel, and M. M.
Caldwell, “Non-invasive measurements of leaf epidermal
transmittance of UV radiation using chlorophyll fluorescence:
field and laboratory studies,” Physiol. Plant. 109, 274 –283
共2000兲.
H. W. Trissl, “Determination of the quenching efficiency of the
oxidized primary donor of Photosystem I, P700⫹: implications for the trapping mechanism,” Photosynth. Res. 54, 237–
240 共1997兲.
Z. G. Cerovic, Y. Goulas, and I. Moya, “Device for measuring
light absorption characteristics of a biological tissue sample,”
Patent WO 03029791 共10 April 2003兲.
G. Samson, O. Prasil, and B. Yaakoubd, “Photochemical and
thermal phases of chlorophyll a fluorescence,” Photosynthetica
37, 163–182 共1999兲.
Z. G. Cerovic, G. Samson, F. Morales, N. Tremblay, and I.
Moya, “Ultraviolet-induced fluorescence for plant monitoring:
present state and prospects,” Agron. Agric. Environ. 19, 543–
578 共1999兲.
A. Cartelat, Z. Cerovic, Y. Goulas, S. Meyer, C. Lelarge, J.-L.
Prioul, A. Barbottin, M.-H. Jeuffroy, P. Gate, G. Agati, and I.
Moya, “Optically assessed content of leaf polyphenolics and
chlorophyll of leaves as indicators of nitrogen deficiency in
wheat 共Triticum aestivum L.兲,” Field Crops Res. 共to be published兲.
A. Ounis, Z. G. Cerovic, J. M. Briantais, and I. Moya, “Dualexcitation FLIDAR for the estimation of epidermal UV absorption in leaves and canopies,” Remote Sens. Environ. 76, 33– 48
共2001兲.
D. S. Ellsworth and P. B. Reich, “Leaf mass per area, nitrogen
content and photosynthetic carbon gain in Acer saccharum
seedlings in contrasting forest light environments,” Funct.
Ecol. 6, 423– 435 共1992兲.
J. Amesz, L. N. M. Duysens, and D. C. Brandt, “Methods for
measuring and correcting the absorption spectrum of scattering suspensions,” J. Theor. Biol. 1, 59 –74 共1961兲.
D. A. Herms and W. J. Mattson, “The dilemma of plants: to
grow or to defend,” Q. Rev. Biol. 67, 283–335 共1992兲.
A. Cartelat, Y. Goulas, C. Lelarge, A. Barbottin, M.-H. Jeuffroy, and Z. G. Cerovic, “A new optical signature of nitrogen
deficiency in wheat useful for decision support in precision
agriculture,” in the Fourth European Conference on Precision
Agriculture, A. Werner and A. Jarfe, eds. 共Wageningen Academic, Berlin, 2003兲, pp. 377–379.