Journal of Experimental Botany, Vol. 66, No. 18 pp. 5595–5603, 2015
doi:10.1093/jxb/erv272 Advance Access publication 12 June 2015
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Estimating chlorophyll content and photochemical yieldof photosystem II (ΦPSII) using solar-induced chlorophyll
fluorescence measurements at different growing stages ofattached leaves
1
2

Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113–8657 Japan
School of Forest Resources, The University of Maine, 5557 Nutting Hall, Orono, ME 04469, USA* To whom correspondence should be addressed. E-mail: aomasa@mail.ecc.u-tokyo.ac.jp
Received 22 November 2014; Revised 7 May 2015; Accepted 11 May 2015
Editor: Tracy Lawson

Abstract
This paper illustrates the possibility of measuring chlorophyll (Chl) content and Chl fluorescence parameters by
the solar-induced Chl fluorescence (SIF) method using the Fraunhofer line depth (FLD) principle, and compares theresults with the standard measurement methods. A high-spectral resolution HR2000+ and an ordinary USB4000
spectrometer were used to measure leaf reflectance under solar and artificial light, respectively, to estimate Chlfluorescence. Using leaves of Capsicum annuum cv. ‘Sven’ (paprika), the relationships between the Chl content andthe steady-state Chl fluorescence near oxygen absorption bands of O2B (686 nm) and O2A (760 nm), measured underartificial and solar light at different growing stages of leaves, were evaluated. The Chl fluorescence yields of ΦF
686 nm/ΦF 760 nm ratios obtained from both methods correlated well with the Chl content (steady-state solar light:
R2 = 0.73; artificial light: R2 = 0.94). The SIF method was less accurate for Chl content estimation when Chl content
was high. The steady-state solar-induced Chl fluorescence yield ratio correlated very well with the artificial-lightinduced one (R2 = 0.84). A new methodology is then presented to estimate photochemical yield of photosystem II(ΦPSII) from the SIF measurements, which was verified against the standard Chl fluorescence measurement method(pulse-amplitude modulated method). The high coefficient of determination (R2 = 0.74) between the ΦPSII of thetwo methods shows that photosynthesis process parameters can be successfully estimated using the presented
methodology.
Key words: Chlorophyll content; different leaf age; Fraunhofer line depth principle; photochemical yield of photosystem II(ΦPSII); solar-induced chlorophyll fluorescence.

Abbreviations: ΦF band, Chl fluorescence yield at O2A or O2B band (nm), where the subscript means the same as that in F; ΦFm’ band, laser saturation pulse-induced
Chl fluorescence yield at O2A band (nm) under solar light; ΦPSII, photochemical yield of photosystem II, where the subscript FLD and PAM mean the value estimated by
the FLD method and that measured by JUNIOR PAM, respectively; λ, wavelength; Chl, chlorophyll; DN, digital number;F band, Chl fluorescence intensity at O2A or O2Bband (nm), where the subscript a and s in F mean artificial-light-induced and solar-induced values, respectively; FLD, Fraunhofer line depth principle; FWHM, full width at
half maximum; LIFT, laser induced fluorescence transients; M, the mean values of radiant intensity (digital number), where the subscript a, b and c, d mean the bands ofborder and bottom of well from non-fluorescent reference and leaf, respectively; O2A, oxygen absorption band of 760 nm; O2B, oxygen absorption band of 686 nm; PAM,
pulse-amplitude modulated; PPF, photosynthetic photon flux (μmol m–2 s–1); PRI, photochemical reflectance index; R, reference coefficient; R2, coefficient of determination;
RMSE, root mean square error; SIF, solar-induced Chl fluorescence; W, wavelength width (nm) at O2A or O2B band shown in Fig.1, the optimum is 0.4 nm.
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Downloaded from http://jxb.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on October 29, 2015

Bayaer Tubuxin1, Parinaz Rahimzadeh-Bajgiran1,2, Yusaku Ginnan1, Fumiki Hosoi1 and Kenji Omasa1*

5596 | Tubuxin et al.

Introduction
stages of the plants when Chl content is variable. In addition, interest in relationships between steady-state SIF andthe photosynthesis reaction at the physiology level, as wellas growth primary production and the enhanced vegetationindex (Frankenberg et al., 2011; Joiner et al., 2011), which are
related to biomass and Chl content, is growing (Van der tol
et al., 2014).
To progress our understanding of the potential to detect
SIF signals from vegetation reflectance to study photosynthesis, the main objective of this paper is to evaluate the performance of the SIF measurement method throughout thelifespan of leaves with varying Chl content through comparison with the standard Chl measurement method. This is carried out via an estimation of Chl content and photochemicalyield of photosystem II (ΦPSII). Not many studies have examined the correlation between both measurement methodsof Chl fluorescence on the same sample. Moya et al. (2004)
found high correlation between both ﬂuorescence measurements; however, the experiments were limited to a small rangeof Chl content (350–450 mg m−2). As far as could be determined, there is currently no report available to estimate andcompare large-range Chl content or ΦPSII obtained from thetwo methods. In the present paper, the focus has, therefore,
been on the relationships between the steady-state Chl fluorescence and Chl content near the oxygen absorption bandsof O2B (686 nm) and O2A (760 nm), measured under artificial light and solar light at different growing stages of leaves.
A new technique to estimate ΦPSII from the SIF method is
also presented.

Materials and methods
Plant material and growth conditions
Paprika (Capsicum annuum cv. ‘Sven’) plants were grown inan environmentally controlled growth chamber for 10–18
weeks. The plants were illuminated for 12 h each day with fluorescent lights and halogen lamps at a photosynthetic photon
flux (PPF) of 400 μmol m–2 s–1. The growth chamber air temperature was 25.0°C during the day and 20.0°C at night withrelative humidity of ~70%. Plants were watered daily witha nutrient solution (1:1000 dilution of HYPONeX). Fully
expanded mature and aging leaves of different growth stages
were used in the experiments.

Chl fluorescence measurement under artificial light
Comparable veinless sites of attached leaves were used to measure the steady-state artificial-light-induced Chl fluorescence.
The fluorescence spectra of leaves were measured at an interval of 500 ms (integration time) with a USB4000 spectrometer(Ocean Optics, USA) under a halogen light (Sumita Optical
Glass, Inc., LS-100F) through short-pass (Optical Coatings
Japan, wavelength λ < 600 nm) and heat-absorbing filters ina dark room. The USB4000 spectrometer was set to the 350–
850 nm range with 3648 channels, grating of 600 lines mm−1,

Downloaded from http://jxb.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on October 29, 2015

Chlorophyll (Chl) fluorescence has been studied as a useful probe for photosynthesis research (Govindjee, 1995,
Papageorgiou and Govindjee, 2004; Buschmann, 2007; Baker,
2008; Omasa et al., 2009; van der Tol et al., 2009; Kalaji et al.,2012; Murchie and Lawson, 2013. It has been extensively
applied for active phenotyping remote sensing of photosynthetic functioning, and for studying biotic and abiotic stress inplants (Omasa, 1998; Omasa et al., 1987, 2007; Lichtenthaler
et al., 1992; Kolber et al., 1998; Kim et al., 2001; Omasa and
Takayama, 2003; Schreiber, 2004; Moya et al., 2004; Konishi
et al., 2009; Pieruschka et al., 2012; 2014).
However, the common active remote sensing method
might be limited to short-distance use for individual leaves
and small plants. Efforts have been made to expand the application of active Chl fluorescence measurement methods forlong-distance, canopy-scale studies using the laser induced
fluorescence transient (LIFT) method. The LIFT approach
employs low-intensity pulses instead of a saturating pulse tomeasure the ﬂuorescence transient, which is interpolated to a
maximum ﬂuorescence level using a ﬂuorescence model. The
technique is capable of measuring Chl fluorescence up to a
distance of 50 m but there are still challenges for more longdistance remote sensing (Kolber et al. 2005; Pierurschka et al.
2012; 2014).
Passive remote sensing of steady-state solar-induced
Chl fluorescence (SIF) has more recently become available(Meroni et al., 2009). The advantages of the SIF method
are that the technique does not require an artificial excitation source and it can be used on air-borne and space-borne
devices for canopy and larger-scale applications. However,this technique requires instruments with high spectral resolution and high accuracy to be able to differentiate the weakfluorescence signal from the strong background light reflection (Meroni and Colombo, 2006).
SIF measurement techniques have been developed forfield and air-borne applications in recent years (Plascyk
et al., 1975; Moya et al., 2004; Liu et al., 2005; Corp et al.,2006; Meroni et al., 2006, 2009; Rascher et al., 2008; Liu andCheng, 2010; Zarco-Tejada et al., 2012). The development of
space-borne SIF remote sensing of GOSAT satellite has provided the opportunity to assess global terrestrial carbon cycle
and has attracted a considerable amount of attention withinscientific communities (Frankenberg et al., 2011; Joiner et al.,2011; Porcar-Castel et al., 2014).
Current trends of research in the SIF field demonstrate
the need for further evaluations of this technique. There are
intrinsic differences between the basis of active Chl fluorescence measurement methods based on pulse-amplitude
modulated (PAM) measurement and that of the SIF; thus,
consequent challenges are currently encountered. Further,leaf-level experimental work is needed to characterize relationships between SIF- and PAM-measured Chl fluorescence(Porcar-Castel et al., 2014). The steady-state Chl fluorescence has been shown to be partially re-absorbed by Chl(Lichtenthaler and Rinderle, 1988; Buschmann, 2007), which
necessitates the evaluation of Chl fluorescence at various lifeEstimating chlorophyll content and ΦPSII using solar-induced chlorophyll fluorescence | 5597

Spectral radiant intensity measurement for chlorophyll
content estimation under solar light using the FLD
methodAfter the above-mentioned artificial-light-induced measurements, the spectral radiant intensity of the same leaf area
was measured at an interval of 200 ms (integration time)
under solar light using a 0.035 nm FWHM HR2000+ spectrometer (Ocean Optics, USA) in the 680–770 nm range with
2048 channels. The spectrometer was built with grating H11
of 1800 lines/mm, a 5 μm slit, an L2 detector collection lens,an OF1-OG590 long-pass filter, and a set of high-reflectivity
AgPlus mirrors model SAG+UPGD-HR. The spectrometer
was connected to a 2 m-long fibre optic, bundled 600 μm optical fibre up, with a CC-3 VIS-NIR cosine corrector-diffuser
for spectral radiant intensity measurement. The attachedleaf was set vertically to incident solar light after sufficient
light adaptation. The fibre-optic diffuser was set at 5 cm distance from the leaf surface, and it was held at 45° to light toreduce specular reflection from the leaf surface. An 18% Gray
Card (Kodak, USA) set at the same angle and position as
the leaf was used as the non-fluorescent reference standard.
Both the spectra of the leaf and the non-fluorescent reference
were recorded to computer via a USB port using OPwave+
operating software (Ocean Optics, USA). Black wool paper
was set at the back of the leaf to remove the reflection oflight transmitted through the leaf. During the experiments,a LI-COR LI-250 light meter was used to measure the PPF(800–1800 μmol m–2 s–1).

Spectral radiant intensity measurement for ΦPSII
estimation under solar light
The attached leaf was set vertically to incident solar lightusing a JUNIOR-B leaf clip (Walz). The fibre-optic diffuserof the HR2000+ spectrometer was set at 5 mm distance fromthe leaf surface, and it was held at 45° to solar light. During
the experiment, the spectral leaf radiant intensity was continuously measured at an interval of 200 ms (integration time)
under solar light using the spectrometer after sufficient light
adaptation. The saturation light pulse of about 5000 μmol
m–2 s–1 from a red laser (KaLaser: 660 nm, 200 mW) was

illuminated for 1 s at 60° to the leaf from a distance of 40 cm
and at about a 30 mm diameter footprint. For the verification, the photochemical yield of photosystem II (ΦPSII) was
measured at a 1 mm distance from the leaf by JUNIOR PAM(about 10000 μmol m–2 s–1 blue LED saturation pulse for 0.6 s
and measuring beam of 5 Hz) to give ΦPSII, PAM. The spectral
radiant intensity data were recorded by the above-mentioned
computer system. The set of sequential experiments included(i) measurement of solar light radiant intensity reflected froma non-fluorescent reference (90% White Card), (ii) measurement of solar light radiant intensity from the attached leaf,(iii) measurement of saturation laser pulse radiant intensityfrom the leaf, (iv) measurement of ΦPSII, PAM of the leaf using
JUNIOR PAM, (v) measurement of solar light radiant intensity from the leaf, (vi) measurement of the second laser pulse
radiant intensity from the leaf, and (vii) measurement ofsolar light radiant intensity reflected from the non-fluorescentreference at each interval of 20 s in (ii) to (vi) and about 60
s in (i) and (vii) under solar light. A data set with very littlechange in solar light intensity during the experiment was used
for the calculation of ΦPSII estimated by the Fraunhofer line
depth principle (FLD), designated ΦPSII, FLD. The solar lightintensity in the experiment was 200–1600 μmol m−2 s−1.

Solar-induced Chl fluorescence estimation using FLD
It is difficult to quantify the SIF from the ordinary spectral leaf radiant intensity because the signal is obscured by
the reflected light. However, the leaf radiant intensity in
Fraunhofer lines increases the ratio of Chl fluorescence emission to reflected light in the leaf radiant intensity as comparedwith other wavelengths, hence the fluorescence emission canbe quantified by the FLD (Lichtenthaler et al., 1992; Moyaet al., 2004).
The FLD method was originally proposed by Plasccyk
and Gabriel (1975), then successfully used by Meroni et al.
(2006; 2008a) and Rascher et al. (2009) at the leaf level, andat a canopy level by Moya et al. (2004), Meroni et al. (2008b),
and Daumard et al. (2012). At the ground level, two oxygen
absorption bands of terrestrial atmosphere at 760 nm (O2A)
and 686 nm (O2B) are usually used to estimate the steadystate SIF because the absorption bands include Chl fluorescence emission spectra.
Fig. 1 shows the FLD method. In Fig. 1A, B, the solid line is
the non-fluorescent reference radiant intensity spectra and thedashed line is the leaf radiant intensity spectra measured in O2A
and O2B bands. Ma and Mb are the mean values of radiant intensity (digital numbers, which is not calibrated to exact radiance)from the non-fluorescent reference in band a (border of thewell, A: W = 685.93 to 686.32 nm; B: W = 758.76 to 759.17 nm;
W: the wavelength width) and in band b (bottom of the well,
A: W = 686.54 to 686.93 nm; B: W = 760.24 to 760.64 nm), andMc and Md are the mean values of radiant intensity from theleaf in band c (the same W as a) and in band d (the same Was b), respectively. Following Plascyk and Gabriel (1975) andMoya et al. (2004), the reflectance coefficient (R) and the solarinduced fluorescence intensity (Fs) were derived from the measured values of Ma, Mb, Mc and Md as follows:

Downloaded from http://jxb.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on October 29, 2015

and a 50 μm slit. It was connected to a 3 m-long fibre cosine
corrector-diffuser. The instrument has 1.5–2.3 nm full widthat half maximum (FWHM). The attached leaf was set at a
45° angle to the optical light beam and the fibre optic diffuser
was set at 5 cm distance from the leaf surface. The fibre-optic
diffuser was equipped with a long-pass filter (Optical Coatings
Japan, λ > 640 nm) to block the reflected light from the leaf.
The LS-1-CAL calibrated tungsten halogen NIST-traceable
light source (Ocean Optics, USA) was used to calibrate thespectrometer. The leaf area was illuminated for 20 min (PPF:
150 μmol m–2 s–1), and then the steady-state Chl fluorescence
was measured. Black wool paper was set at the back of theleaf to remove the reflection of light transmitted through theleaf. Chl fluorescence spectra were recorded to computer via a
USB port using SpectraSuite operating software.

5598 | Tubuxin et al.
in the SIF yield (ΦF760.4) and laser saturation pulse-induced
yield (ΦFm’760.4) of the O2A band were calculated by the
FLD method. ΦPSII, FLD was calculated by
Φ PSII, FLD = ( ΦFm’ − ΦFs ) / ΦFm’

(4)

where ΦFs is a constant value after median filtering to datameasured during the 10 s just before the saturation laser pulse
for noise reduction, and ΦFm’ is the mean value of the highest three data points during the saturation laser pulse.

R = (Mc − Md ) / (Ma − Mb )(1)

Fs = Md – R × Mb

(2)When the saturation laser pulse-induced fluorescence intensity under solar light is calculated by equations (1) and (2),the radiant intensity Ma and Mb from the non-fluorescentreference under only solar light are used because the radiantintensity in a and b bands of O2A is not affected by the redlaser pulse. The radiant intensity Mc and Md apply to thosefrom the leaf under both solar and laser lights.

Chl fluorescence yield ratio
The Chl fluorescence yield (ΦF, the ratio of fluorescence photons to absorbed photons) can more accurately represent therelationship between the Chl fluorescence signal magnitude
and light intensity than Chl fluorescence intensity (Govindjee,1995). The solar-induced and artificial-light-induced Chlfluorescence yields near the 686 nm and 760 nm bands wereapproximately calculated by
ΦFs = F / ( PPF × 0.84 )(3)

where F is the fluorescence intensity (relative units), PPF is thephotosynthetic photon flux (μmol m–2 s–1), and 0.84 is the leaf
absorption coefficient. The solar-induced (ΦFs) and artificiallight-induced (ΦFa) Chl fluorescence yield ratios of ΦFs686.7/
ΦFs760.4 and ΦFa686.4/ΦFa760.5 were calculated and theirrelationships at different Chl content levels compared. The
ratio of [ΦFs686.7/ΦFs760.4]/[ΦFa686.4/ΦFa760.5] was thencalculated to assess the steady-state SIF at different leaf-growing stages. The wavelength width (W in Fig. 1) to minimize themean absolute error of SIF estimation was 0.4 nm.

ΦPSII, FLD estimation from solar-induced and red laser
saturation pulse-induced Chl fluorescence yields
From a set of temporal changes in the above-mentioned spectral radiant intensity measurement of (1) to (7), the changes

Leaf spectral absorptance
Chl fluorescence spectra largely overlap with Chl absorption,and the red fluorescence re-absorbed by Chl itself is larger
than the far-red fluorescence (Govindjee, 1995; Gitelson
et al. 1998). To investigate the re-absorption in Chl fluorescence spectra, the reflectance (R) and transmittance (T) of
the leaf were measured using a spectrophotometer equippedwith an integrating sphere (V570, JASCO, Japan) in a rangeof 600–800 nm and a spectral resolution of 2 nm. The reflectance spectra were measured against barium sulphate as a reference standard; black wool paper was set at the back of theleaf to remove the re-reflection of light transmitted throughthe leaf. Leaf spectral absorptance was calculated as 1-T-R(Buschmann et al., 1994; Gitelson et al., 1998, 2003).

Chlorophyll content
A 1.5 cm-diameter leaf disc was cut from the marked pointof the leaf using a standard leaf punch. After weighing, theleaf disc was mashed in 96% ethanol solution using a mortar and a pestle. The pigments were completely separated in
sedimentation tubes by a centrifugal separator set for 5 min
at 2000 rpm. The absorption spectrum of the top layer solution in the tube was measured by a V570 spectrophotometer (JASCO, Japan) at 0.5 nm intervals between 600 nm and
800 nm. Total Chl content was calculated from the absorption spectra using equations reported by Wintermans and deMots (1965).

Results
Figure 2A shows the steady-state Chl fluorescence emission
spectrum of three leaves with different Chl content taken
under the artificial illumination light (λ < 600 nm). Two maxima appeared in the red region around 686 nm (F686) and
far-red region around 740 nm (F740). Near the red region,
Chl fluorescence increased sharply with the reduction of Chlcontent, but near the far-red region, it increased with theincrease of Chl content. The Chl absorptance near 686 nm
greatly increased with increasing Chl content, whereas there
was little difference near 760 nm (Fig. 2B).
Figure 3 shows relationships between leaf Chl content
and steady-state Chl fluorescence yields of ΦFa686.4 andΦFa760.5 under artificial light. The Chl fluorescence yields ofΦFa686.4 decreased with an increase in Chl content (Fig. 3A),
whereas those of ΦFa760.5 increased (Fig. 3B), although theyields greatly varied at low Chl content below about 200 mg

Downloaded from http://jxb.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on October 29, 2015

Fig. 1. Concept of Fraunhofer line depth (FLD) method. (A) O2B band.
(B) O2A band. The solid line is non-fluorescent reference radiant intensity
spectra and the dashed line is paprika (Capsicum annuum cv. ‘Sven’) leaf
radiant intensity spectra measured in O2A and O2B bands. The bands aand c are the border of the well and the bands b and d are the bottom of
the well for the reflectance and leaf. The W is wavelength width in band a,b, c, and d. The measured value of radiant intensity is shown as the digital
number (DN) and is not calibrated to exact radiance. The leaf Chl content
was 621.1 mg m−2.

Estimating chlorophyll content and ΦPSII using solar-induced chlorophyll fluorescence | 5599
Figs. 3 and 4. Under artificial light, the ratio of ΦFa686.4/
ΦFa760.5 showed a very clearly inverse curvilinear relationship with the Chl content of leaves (Fig. 5A; R2 = 0.94).
A similar highly inverse curvilinear relationship was also
obtained under solar light (Fig. 5B; R2 = 0.73) regardless of
the large variation shown in Fig. 4. The lower coefficient ofdetermination value obtained for the solar light-induced yieldratio is caused by wider variation at all Chl content values.
A comparison of root mean square error (RMSE) values as
indicated on the figure shows a much larger RMSE for the
SIF method. However, when RMSEs are calculated for Chlcontent values below 400 mg m−2, the RMSEs of the two
methods become closer and amount to a maximum of 40 mg
m−2 for the SIF method, indicating that the SIF method
might be less accurate for higher Chl content (above 400 mgm−2) estimation.
Figure 6 shows a linear correlation between artificial-lightinduced Chl fluorescence yield ratio (ΦFa686.4/ΦFa760.5)
and the solar-induced one (ΦFs686.7/ΦFs760.4) obtained at
the optimal W (W = 0.4nm). The regression line was very
close to the y = x line.
Figure 7 shows an example of Chl fluorescence measurement using the FLD method under solar light and saturation
pulse for estimating ΦPSII of the attached leaf. The temporal changes in radiant intensity measurement of (ii) to (vi)Fig. 2. Steady-state Chl fluorescence emission spectra and absorption
spectra of different Chl content leaves taken under artificial illumination (λ< 600 nm). (A) The steady-state Chl fluorescence emission spectra. (B)Absorptance spectra of the same leaf sample. Chl content: the solid line
is 502.3 mg m−2, the dashed line is 160.8 mg m−2, and the dotted line is62.9 mg m−2.

Fig. 5. Relationships between leaf Chl content and steady-state Chlfluorescence yield ratios calculated from Figs. 3 and 4, respectively. (A)Artificial-light-induced Chl fluorescence yield ratio (ΦFa686.4/ΦFa760.5). (B)SIF yield ratio (ΦFs686.7/ΦFs760.4).

Fig. 3. Relationships between leaf Chl content and steady-state artificiallight-induced Chl fluorescence yields. (A) ΦFa686.4. (B) ΦFa760.5.

Fig. 4. Relationships between leaf Chl content and steady-state SIF
yields. (A) ΦFa686.7. (B) ΦFa760.4.

Fig. 6. Relationship between artificial-light-induced Chl fluorescence
yield ratio (ΦFa686.4/ΦFa760.5) and solar-induced yield ratio (ΦFs686.7/
ΦFs760.4) at different growing stages of paprika leaves.

Downloaded from http://jxb.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on October 29, 2015

m−2. At higher Chl content with more than about 400 mg m−2,
the changes in ΦFa686.4 and ΦFa760.5 become small.
Figure 4 shows the relationships between leaf Chl content and steady-state solar-induced Chl fluorescence yieldsof ΦFs686.7 and ΦFs760.4. The Chl fluorescence yields ofΦFs686.7 decreased with an increase in Chl content (Fig. 4A),
whereas the Chl fluorescence yields of ΦFs760.4 increased,
although the yields varied largely, especially in ΦFs760.4(Fig. 4B). Comparing Figs. 3 and 4, the solar-induced Chlfluorescence yield variation was larger than that induced byartificial light, probably because of a weak Chl fluorescencesignal, lack of the sensitivity of the spectrometer, and solar
fluctuation during the measurement.
Figure 5 shows the relationships between leaf Chl content
and steady-state Chl fluorescence yield ratios calculated from

5600 | Tubuxin et al.
Figure 8 shows a linear correlation between ΦPSII, PAM(measured by JUNIOR PAM) and ΦPSII, FLD (estimated by
FLD method) at different growing stages of the attachedleaves. The regression line was approximated by y = x.

Discussion

mentioned in Materials and methods, Spectral radiant intensity measurement for ΦPSII estimation under solar light, areshown in Fig. 7A. The radiant intensity increased with the saturation pulses of 660 nm laser (SLRL) and blue LED (SLPAM)
of JUNIOR PAM. Fig. 7B, C shows temporal changes in
Chl fluorescence intensity (F760.4) calculated by the FLD
method and those in Chl fluorescence yield (ΦF760.4) calculated by equation (3). The solid line in Fig. 7C is the sunlightfluorescence yield (ΦF) without noise after median filtering during 10 s before the SLRL illumination for estimating
ΦPSII. The saturation laser-induced fluorescence yield (ΦFm’)
under solar light was larger than the yield induced by SLPAM.
This result might have been caused by the difference in beamfootprint sizes between the HR2000+ spectrometer and
JUNIOR PAM.

Downloaded from http://jxb.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on October 29, 2015

Fig. 7. Measurement of Chl fluorescence using the FLD method undersolar light and saturation pulse for estimating ΦPSII of paprika leaf. (A)Temporal changes in leaf radiant intensity spectra measured using a
HR2000+ spectrometer under solar light (1450 μmol m−2 s−1), where SLRL
is the saturation light pulse (4450 μmol m−2 s−1) using red laser (660 nm)
and SLPAM is the saturation light pulse (blue LED, about 10 000 μmol m−2s−1) of JUNIOR PAM. The solid line is the spectra of 758.9 nm and thedotted line is that of 760.4 nm. A mean value of 0.4 nm wavelength width(W) was used. (B) Temporal changes in Chl fluorescence intensity (F760.4)
calculated by the FLD method. (C) Temporal changes in Chl fluorescence
yield (ΦF760.4). The dotted line is the changes in ΦF760.4 and the solidline is the value after median filtering during the 10 s before the SLRL
illumination for estimating ΦPSII.

Active Chl fluorescence analysis such as the PAM method toassess photosynthesis activities is the most common method
employed for laboratory-scale photosynthesis studies andcan provide invaluable information on the factors affecting
the photosynthesis process. However, this method has limitations for large-size plants or at canopy level. For this reason,
the passive remote sensing of photosynthesis can be morefeasibly carried out either through SIF studies or the photochemical reflectance index (PRI) measurement introduced
by Gamon et al. (1990). Although PRI has been widely used
for photosynthesis measurement, recent studies have shownthat PRI use may be limited by its sensitivity to plant pigment
variation at various life stages of the leaves (RahimzadehBajgiran et al., 2012).
A few studies have been recently performed on the solarinduced Chl fluorescence at both laboratory scale and canopy level for plant photosynthesis measurement (Moya et al.
2004; Meroni et al., 2006; 2009; Rascher et al., 2008). Under
solar light, the steady-state Chl fluorescence cannot be measured directly, because the photon emission from the canopy isless than 1% of what is emitted by the sun (Liu et al., 2005).
Hence, the SIF measurement using Fraunhofer line spectrasuch as the FLD principle has been proposed as the only way
to remotely sense Chl fluorescence in natural environments.
As outlined by others (Malenovský et al., 2009; Porcar-Castel
et al., 2014; Van der Tol et al., 2014), however, there is a needfor further studies relating the steady-state SIF measurements
to photosynthetic performance of plants.
The result presented here show that the steady-state Chlfluorescence emission spectrum in the red peak near 686 nm
strongly overlaps with the maximum of the leaf absorption
spectrum near 680 nm (Fig. 2). At the red region near O2B(686nm), the steady-state Chl fluorescence yield decreased
sharply with the increase of Chl content regardless of thetype of the light source-artificial or solar (Figs. 3A and 4A).
This is speculated to be due to the fact that a large part of the
red Chl fluorescence is re-absorbed by the Chl pigment before
it can be measured. At the far-red region near O2B (760nm),
because of the sharp decrease of the absorption of the leaf,
the Chl fluorescence yield rose with Chl content increase(Figs. 3B and 4B). The phenomenon that Chl fluorescenceis strongly re-absorbed by Chl pigment near the red region
more than the far-red region has previously been reported(Lichtenthaler et al., 1986; Gitelson et al., 1998; Buschmann,2007). The present results obtained by the FLD principle
using O2A and O2B bands followed the same phenomenon.
Under solar light, the Chl fluorescence signal largely overlapped with the high-reflection signal of incident solar lightand the re-absorption bands of pigments, and it is easily
affected by the instrument conditions, the intensity of exciting
light, and the angle of detection. The measurement of O2A

Estimating chlorophyll content and ΦPSII using solar-induced chlorophyll fluorescence | 5601
At the band width of 0.4 nm, the steady-state Chl fluorescence yield ratios of the two methods were very well correlated with an R2 of 0.84 (Fig. 6). The work by Moya et al.
(2004) reported a high correlation between active and passive fluorescence measurements (R2 = 0.99) (not the ratio, justabsolute values), but this was obtained over a limited range of
Chl content, eliminating the effects of re-absorption.
The saturation pulse method has been proposed as a powerful tool for assessing photosynthetic parameters (Genty
et al., 1989; Bilger and Björkman, 1990; Schreiber 2004) andmany useful Chl fluorescence parameters, such as ΦPSII and
non-photochemical quenching, have been developed andused as indices of photosynthetic activity under solar andartificial (actinic) lights. The value obtained by multiplying
the PPF value of actinic light by the ФPSII value is used as anindicator of photosynthetic electron transport rate and CO2
fixation rate (Baker and Oxborough, 2004; Schreiber, 2004).
The PAM Chl fluorometer is the standard instrument forthe measurement of the Chl fluorescence parameters using
the saturation pulse method. Therefore, the Chl fluorescenceparameter ΦPSII,FLD estimated using the FLD method undersolar light and saturation pulse was verified with a PAM
Chl fluorometer (JUNIOR PAM) according to the sequence
shown in Fig. 7. Although the Chl fluorescence signal estimated by the FLD method was very noisy, the stable ΦFs760.4
and ΦFm’760.4 in equation (4) for calculating ΦPSII, FLD wereobtained as a constant value by the median filtering fromdata measured during the 10 s just before the saturation laser
pulse (SLRL) and averaging of the highest three data points
during the saturation laser pulse. To assure the reliability in
ΦPSII, FLD estimation, the small change in solar intensity wasconfirmed during a sequence from (i) to (vii) in the measurement mentioned in the Materials and methods. As a result,the ΦFs760.4 and ΦFm’760.4 values were stable during double measurements by the saturation laser pulse. Though the
JUNIOR PAM blue LED saturation pulse (SLPAM) during
0.6 s was illuminated on the leaf at about 10000 μmol m–2
s–1, the Chl fluorescence yield estimated by the FLD method
hardly changed (Fig. 7C). This could be owing to the measured error of SLPAM (Fig. 7A), caused by the difference inbeam footprint size between the HR2000+ spectrometer and
JUNIOR PAM.
The ΦPSII, FLD and ΦPSII, PAM exhibited a good correlation
with R2 of 0.74 and RMSE of 0.08, regardless of different
leaf ages and solar intensities of 200–1600 μmol m−2 s−1(Fig. 8). The regression line was almost y = x. This result istherefore introduced as a newly developed ΦPSII measurement
technique using the FLD method. Potential sources of error
may include noise in the spectrometer, the solar fluctuation
during the measurement, differences in the angle of the uneven leaf surface and non-fluorescent reference, the accuracyof light intensity measurement on the leaf, etc. These should
be taken into consideration when trying to develop specificinstruments designed based on the findings presented here.
Should the remote sensing of ΦPSII for whole-plant andcanopy scale studies using the SIF method be desired, ahigh-power laser or LED for the saturation pulse would beneeded. This is a limitation of the saturation pulse method,

Downloaded from http://jxb.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on October 29, 2015

and O2B reflectance under solar light is very difficult and one
should be careful to provide identical illumination to the sample and the reference, because the depth of oxygen absorption
bands may change easily (Zarco-Tejada et al., 2003; Moyaet al., 2004; Meroni et al., 2006; Campbell et al., 2008). In thisstudy, the reflectance of the standard reflectance panel wasmeasured immediately after the measurement of the sample reflectance, but changes in the oxygen absorption bands
leading to measurement errors are inherently inevitable.
Also, under the unstable solar light intensity, the instabilityof photosynthetic states results in a change in the percentage
of Chl fluorescence emission and Chl re-absorption capacity(Buschmann, 2007).
In practice, the Chl fluorescence intensity ratio of F685/
F730, F690/F735, or F690/F740 is used to assess the steadystate artificial-light-induced Chl fluorescence signals anddetermine the Chl content in a non-destructive way (Agatiet al. 1995; Buschmann, 2007; Campbell et al., 2008).
Gitelson et al. (1998) pointed out that ≥90% of the variationin the fluorescence ratio F690/F735 is exclusively dominatedby Chl content. In contrast, at the ground level, two oxygen
absorption bands of terrestrial atmosphere at 686 nm (O2B)
and 760 nm (O2A) are usually used to estimate steady-state
SIF because the position of the absorption bands are very
close to the position of the Chl fluorescence spectrum peaks(Moya et al., 2004; Meroni et al., 2009). Here, the Chl fluorescence yield ratio of O2A and O2B was used to estimate thechange of steady-state Chl fluorescence with Chl content andwas considered to better express the relationship between the
Chl fluorescence and illumination light.
The steady-state artificial-light-induced Chl fluorescence
yield ratio of ΦFa686.4/ΦFa760.5 and the steady-state SIF
yield ratio of ΦFs686.7/ΦFs760.4 showed very clear inverse
curvilinear correlations with Chl content with R2 values of
0.94 and 0.73, respectively (Fig. 5). Moya et al. (1992) mentioned that a strong inverse relationship existed between fluorescence quantum yield and Chl content. Owing to the strong
re-absorption influence of Chl pigment for Chl fluorescencenear the 686 nm band, the Chl fluorescence yield ratios ofΦFa686.4/ΦFa760.5 and ΦFs686.7 /ΦFs760.4 decreased with
the increase of Chl content. Similar trends and shapes of thecurves can be observed in Fig. 5A, B. However, the coefficients of regression lines are different. This is solely related to
the lower accuracy of either measurement method to estimate
Chl content at higher Chl content values. Up to a Chl content
of about 300 mg m−2, the coefficients of the power law functions are very similar with close R2 values (y = 223.2x−0.851,
R2 = 0.80 for Fig. 5A; y = 241.4x−0.867, R2 = 0.79 for Fig. 5B).
Once data pertaining to higher Chl content are included, thecurves start to divert from each other, causing considerable
differences in coefficients. This shows that both the active andpassive methods used here have the same limitation at higher
Chl content: under solar light, when the Chl content is very
high (more than 400 mg m−2), the value of ΦFs686.7/ΦFs760.4
becomes disperse; it appears that sometimes the Chl fluorescence yield ratio is difficult to estimate because of the too-low
fluorescence intensity due to the high re-absorption of Chl
pigment (Fig. 5B).

5602 | Tubuxin et al.

but using the special sequence of laser illumination employing low-intensity pulses as described in Kolber et al. (1998),
it will be possible to interpolate to a maximum ﬂuorescencelevel. Therefore, there is the possibility for remote sensing of
Chl fluorescence parameters such as ΦPSII without the useof accurate pulse-synchronized and modulated fluorimetrictechniques such as PAM and LIFT using the SIF method andthe illumination technique together.
Based on what was discussed above, the SIF measurement method could be used for both the Chl content estimation and photosynthesis parameter measurement (ΦPSII)
needed for phenotyping studies of plants and remote sensing
at different scales. In general, the SIF (FLD) method needs
a high-spectral resolution and a high-sensitivity low-noise
spectrometer to perform well. With more developments in
instrument design and capability expected, the SIF method
could potentially work well and perform more effectively
should more research be focused on the development of spectrometers. For instance, if a cooled high-spectral resolution
spectrometer with high sensitivity and low noise is used, morestable and low-noise data could be obtained.

Conclusion
This paper illustrates the possibility of measuring Chl content
and Chl fluorescence parameters using SIF measurement (FLD
method) and the saturation pulse method via a red laser at different life stages of plant leaves. The results were compared withthose obtained through the standard Chl fluorescence measurement method. A high-resolution HR2000+ spectrometer wasused to measure leaf reflectance under solar light using the
FLD method to estimate the solar-induced Chl fluorescence.
A USB4000 spectrometer measured the steady-state artificiallyinduced Chl fluorescence spectrum in laboratory conditions.
The steady-state SIF yield ratio of O2A and O2B showed
high correlation with Chl content but the relationship was
weaker when comparing with results obtained using artificial
light. Both methods proved to be less accurate at higher Chlcontent owing to the strong re-absorption influence of Chl
pigment for Chl fluorescence, with the SIF method being less

References
Agati G, Mazzinghi P, Fusi F, Ambrosini I. 1995. The F685/F730
chlorophyll fluorescence ratio as a tool in plant physiology: response tophysiological and environmental factors. Journal of Plant Physiology 145,
228–238.
Baker NR. 2008. Chlorophyll fluorescence:aA probe of photosynthesis invivo. Annual Review of Plant Biology. 59, 89−113.
Baker NR, Oxborough K. 2004.Chlorophyll fluorescence as probeof photosynthetic productivity. In: Papageorgiou GC, Govindjee, eds.
Chlorophyll a Fluorescence: A Signature of Photosynthesis. Advances in
Photosynthesis and Respiration, vol. 19. Dordrecht: Springer; 65–82.
Bilger W, Björkman O. 1990. Role of the xanthophyll cycle inphotoprotection elucidated by measurements of light-induced absorbencychanges, fluorescence and photosynthesis in leaves of Hedera
canariensis. Photosynthesis Research 25, 173–185.
Buschmann C, Nagel E, Szabo K, Kocsanyi L. 1994. Spectrometer forfast measurements of in vivo reflection, absorption and fluorescence in thevisible and near infrared. Remote Sensing of Environment. 48, 18−24.
Buschmann C. 2007. Variability and application of chlorophyll fluorescenceemission ratio red/far-red of leaves. Photosynthesis Research 92, 261−271.
Campbell PKE, Middleton EM, Corp LA, Kim MS. 2008. Contributionof chlorophyll fluorescence to the apparent vegetation reflectance. Scienceof the Total Environment 404, 433−439.
Corp LA, Middleton EM, McMurtrey JE, Campbell PKE, Butcher
LM. 2006. Fluorescence sensing techniques for vegetation assessment.
Applied Optics 45, 1023–1033.
Daumard F, Goulas Y, Champagne S, Fournier A, Ounis A, Olioso
A, Moya I. 2012. Continuous monitoring of canopy level sun-induced
chlorophyll fluorescence during the growth of a sorghum field. IEEE
Transactions on Geoscience and Remote Sensing 50, 4292–4300.
Frankenberg C, Fisher JB, Worden J, Badgley G, Saatchi SS, Lee
J-E, Toon GC, Butz A, Jung M, Kuze A, Yokota T. 2011. New global
observations of the terrestrial carbon cycle from GOSAT: patterns ofplant fluorescence with gross primary productivity. Geophysical Research
Letters 38, L17706.
Gamon JA, Field CB, Bilger W, Bjorkman O, Fredeen AL, Penuelas
J. 1990. Remote-sensing of the xanthophyll cycle and chlorophyll
ﬂuorescence in sunﬂower leaves and canopies. Oecologia 85, 1−7.
Genty B, Briantais JM, Baker NR. 1989. The relationship between
the quantum yield of photosynthetic electron-transport and quenching
chlorophyll fluorescence. Biochimica et Biophysica Acta 990, 87–92.
Gitelson AA, Buschmann C, Lichtenthaler HK. 1998. Leaf chlorophyll
fluorescence corrected for re-absorption by means of absorption andreflectance measurements. Journal of Plant Physiology 152, 283−296.

Downloaded from http://jxb.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on October 29, 2015

Fig. 8. Relationship between ΦPSII, PAM and ΦPSII, FLD at different growing
stages of paprika leaves. The SLRL was about 5000 μmol m−2 s−1 and thesolar light intensity was 200–1600 μmol m−2 s−1.

accurate than the artificial-light-induced method. The steadystate SIF yield ratio and the steady-state artificial-lightinduced Chl fluorescence yield ratio were very well correlated(R2 = 0.84), indicating that Chl content can be estimated fromthe proposed SIF measurement method.
A methodology was presented to estimate ΦPSII from the SIF
measurements, and verified against the active Chl fluorescencemethod (PAM). The high coefficient of determination foundbetween ΦPSII of the two methods shows that photosynthesis process parameters can be successfully estimated using the presented
methodology. This can be the basis for future developments inremote sensing technologies for the estimation of both Chl content and Chl fluorescence parameters for passive phenotyping.
To scale-up the measurement techniques using SIF, measures should also be taken to remove the effect of noise, suchas high background radiation, terrestrial atmosphere influence, and instrument measurement errors. Further studies
are also recommended to evaluate these relationships beyond
single leaf studies toward heterogeneous canopies.

Estimating chlorophyll content and ΦPSII using solar-induced chlorophyll fluorescence | 5603

Liu LY, Cheng ZH. 2010. Detection of vegetation light-use efficiency
based on solar-induced chlorophyll fluorescence separated from canopy
radiance spectrum. IEEE J–STARS 3, 306−312.
Malenovský Z, Mishra KB, Zemek F, Rascher U, Nedbal L. 2009.
Scientific and technical challenges in remote sensing of plant canopy
reflectance and fluorescence. Journal of Experimental Botany 60, 2987−3004.

Omasa, K. 1998. Image instrumentation of chlorophyll a fluorescence.
SPIE 3382, 91–99.
Omasa K, Hosoi F, Konishi A. 2007. 3D lidar imaging for detecting
and understanding plant responses and canopy structure. Journal of
Experimental Botany 58, 881−898.
Omasa K, Konishi A, Tamura H, Hosoi F. 2009. 3D confocal laser
scanning microscopy for the analysis of chlorophyll fluorescenceparameters of chloroplasts in intact leaf tissues. Plant and Cell Physiology
50, 90–105.
Omasa K, Takayama K. 2003. Simultaneous measurement of stomatal
conductance, non-photochemical quenching, and photochemical yieldof photosystem II in intact leaves by thermal and chlorophyll fluorescenceimaging. Plant and Cell Physiology 44, 1290−1300.
Omasa K, Shimazaki K, Aiga I, Larcher W, Onoe M. 1987. Image
analysis of chlorophyll fluorescence transients for diagnosing thephotosynthetic system of attached leaves. Plant Physiology 84, 748−752.
Papageorgiou GC, Govindjee eds. 2004. Chlorophyll a Fluorescence:
A Signature of Photosynthesis. Advances in Photosynthesis andRespiration, vol. 19. Dordrecht: Springer.
Plascyk JA, Gabriel FC. 1975. The Fraunhofer Line Discriminator
MK II- an airborne instrument for precise and standardized ecologicalluminescence measurements. IEEE Transactions on Instrumentation andMeasurement 24, 306−313.
Pieruschka R, Klimov D, Berry JA, Osmond CB, Rascher U, Kolber
ZS 2012. Remote chlorophyll fluorescence measurements with the laserinduced fluorescence transient approach. Methods in Molecular Biology
918, 51–59.
Pieruschka R, Albrecht H, Muller O, Berry JA, Klimov D, Kolber
ZS, Malenovský Z, Rascher U. 2014. Daily and seasonal dynamics
of remotely sensed photosynthetic efficiency in tree canopies. Tree
Physiology 34, 674–685.
Porcar-Castell A, Tyystjarvi E, Atherton J, van der Tol C, Flexas
J, Pfundel EE, Moreno J, Frankenberg C, Berry JA. 2014. Linking
chlorophyll a fluorescence to photosynthesis for remote sensing
applications: mechanisms and challenges. Journal of Experimental Botany
65, 4065–4095.
Rahimzadeh-Bajgiran P, Munehiro M, Omasa K. 2012. Relationships
between the photochemical reflectance index (PRI) and chlorophyll
fluorescence parameters and plant pigment indices at different leaf growthstages. Photosynthesis Research 113, 261−271.
Rascher U, Gioli B, Miglietta F. 2008. FLEX-fluorescence explorer:a remote sensing approach to quantify spatio-temporal variations ofphotosynthetic efficiency from space. In: Allen JF, Gantt E, Golbeck
JH, Osmond B, eds. 14th International Congress on Photosynthesis.
Dordrecht: Springer; 1387−1390.

Meroni M, Colombo, R. 2006. Leaf level detection of solar induced
chlorophyll fluorescence by means of a subnanometer resolution
spectroradiometer. Remote Sensing of Environment 103, 438−448.

Rascher U, Agati G, Alonso L, et al. 2009. CEFLES2: the remotesensing component to quantify photosynthetic efficiency from the leaf
to the region by measuring sun-induced fluorescence in the oxygen
absorption bands. Biogeosciences Discussions 6, 2217–2266.

Meroni M, Picchi V, Rossini M, Cogliati S, Panigada C, Nali
C,Lorenzini G, Colombo R. 2008a. Leaf level early assessment of ozone
injuries by passive fluorescence and PRI. International Journal of Remote
Sensing 29, 5409−5422.

Schreiber U. 2004. Pulse-amplitude-modulation (PAM) fluorometry andsaturation pulse method: an overview. In: Papageorgiou GC, Govindjee,eds. Chlorophyll a Fluorescence: A Signature of Photosynthesis. Advancesin Photosynthesis and Respiration, vol. 19. Dordrecht: Springer; 279–319.

Meroni M, Rossini M, Picchi V, Panigada C, Cogliati S, Nali C,
Colombo R. 2008b. Assessing steady-state fluorescence and PRI fromhyperspectral proximal sensing as early indicators of plant stress: the caseof ozone exposure. Sensors 8, 1740−1754.

van der Tol C, Verhoef W, Rosema A. 2009. A model for chlorophyll
fluorescence and photosynthesis at leaf scale. Agricultural and Forest
Meteorology 149, 96–105.

Meroni M, Rossini M, Guanter L, Alonso L, Rascher U, Colombo
R, and Moreno J. 2009. Remote sensing of solar-induced chlorophyll
fluorescence: review of methods and applications. Remote Sensing ofEnvironment 113, 2037–2051.
Moya I, Guyot G, Goulas Y. 1992. Remotely sensed blue and red
fluorescence emission for monitoring vegetation. ISPRS Journal of
Photogrammetry and Remote Sensing 47, 205−231.
Moya I, Camenenb L, Evain S, Goulas Y, Cerovic ZG, Latouche G,
Flexas J, Ounis A. 2004. A new instrument for passive remote sensing:
1. Measurements of sunlight-induced chlorophyll fluorescence. Remote
Sensing of Environment 91, 186−197.
Murchie EH, Lawson T. 2013. Chlorophyll fluorescence analysis: a guideto good practice and understanding some new applications. Journal of
Experimental Botany 64, 3983–3998.

van der Tol C, Berry JA, Campbell PKE, Rascher U. 2014. Models
of ﬂuorescence and photosynthesis for interpreting measurements ofsolar-induced chlorophyll ﬂuorescence, Journal of Geophysical Research:
Biogeoscience 119, 2312–2327.
Wintermans JFGM, de Mots A. 1965. Spectrophotometriccharacteristics of chlorophylls a and b and their pheophytins in ethanol.
Biochimmica et Biophysica Acta 109, 448−453.
Zarco-Tejada PJ, Pushnik JC, Dobrowski S, Ustin SL. 2003.
Steady-state chlorophyll a fluorescence detection from canopy derivativereflectance and double-peak red-edge effects. Remote Sensing ofEnvironment 84, 283–294.
Zarco-Tejada PJ, González–Dugo V, Berni JAJ. 2012. Fluorescence,
temperature and narrow-band indices acquired from a UAV platform forwater stress detection using a micro-hyperspectral imager and a thermal
camera. Remote Sensing of Environment 117, 322–337.

Downloaded from http://jxb.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on October 29, 2015

Gitelson AA, Gritz Y, Merzlyak MN. 2003. Relationships between leaf
chlorophyll content and spectral reflectance and algorithms for nondestructive chlorophyll assessment in higher plant leaves. Journal of Plant
Physiology 160, 271−282.
Govindjee. 1995. Sixty-three years since Kautsky: chlorophyll afluorescence. Australian Journal of Plant Physiology 22, 131–160.
Joiner J, Yoshida Y, Vasilkov A P, Yoshida Y, Corp LA, Middleton
EM. 2011. First observations of global and seasonal terrestrial chlorophyll
fluorescence from space. Biogeosciences 8, 637–651.
Kalaji HM, Goltsev V, Bosa K, Allakhverdiev SI, Strasser RJ. 2012.
Experimental in vivo measurements of light emission in plants: a perspective
dedicated to David Walker. Photosynthesis Research 114, 69−96.
Kim MS, McMurtrey JE, Mulchi CL, Daughtry CST, Chappelle EW,
Chen YR. 2001. Steady-state multispectral fluorescence imaging systemfor plant leaves. Applied Optics 40, 157−166.
Kolber ZS, Prasil O, Falkowski PG. 1998. Measurements of variablechlorophyll ﬂuorescence using fast repetition rate techniques: deﬁning
methodology and experimental protocols. Biochimica et Biophysica Acta
1367, 88–106.
Kolber ZS, Klimov D, Ananyev G, Rascher U, Berry J, Osmond
B. 2005. Measuring photosynthetic parameters at a distance: laser
induced fluorescence transient (LIFT) method for remote measurements ofphotosynthesis in terrestrial vegetation. Photosynthesis Research 84, 121–129.
Konishi A, Eguchi A, Hosoi F, Omasa K. 2009. 3D monitoring spatiotemporal effects of herbicide on a whole plant using combined range andchlorophyll a fluorescence imaging. Functional Plant Biology 36, 874–879.
Lichtenthaler HK, Buschumann C, Rinderle U, Schmuck G. 1986.
Application of chlorophyll fluorescence in ecophysiology. Radiation andEnvironmental Biophysics 25, 297−308.
Lichtenthaler HK, Rinderle U. 1988. The role of chlorophyll fluorescencein the detection of stress conditions in plants. CRC Critical Reviews inAnalytical Chemistry 19, S29–S85.
Lichtenthaler HK, Stober F, Lang M. 1992. The nature of the different
laser-induced fluorescence signatures of plants. EARSeL Advances in
Remote Sensing 1, 20–32.
Liu LY, Zhang YJ, Wang JH, Zhao CJ. 2005. Detecting solar-induced
chlorophyll fluorescence from field radiance spectra based on the
Fraunhofer line principle. IEEE Transactions on Geoscience and Remote
Sensing 43, 827−832.