Plant Cell Physiol. 44(12): 1290–1300 (2003)
JSPP © 2003

Simultaneous Measurement of Stomatal Conductance, Non-photochemical
Quenching, and Photochemical Yield of Photosystem II in Intact Leaves by
Thermal and Chlorophyll Fluorescence Imaging
Kenji Omasa 1 and Kotaro Takayama
Department of Biological and Environmental Engineering, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1
Yayoi, Bunkyo, Tokyo, 113-8675 JapanA new imaging system capable of simultaneously
measuring stomatal conductance and fluorescence parameters, non-photochemical quenching (NPQ) and photochemical yield of photosystem II (FPSII), in intact leaves underaerobic conditions by both thermal imaging and chlorophyll fluorescence imaging was developed. Changes in
distributions of stomatal conductance and fluorescenceparameters across Phaseolus vulgaris L. leaves induced by
abscisic acid treatment were analyzed. A decrease in stomatal conductance expanded in all directions from thetreatment site, then mainly spread along the lateral vein
toward the leaf edge, depending on the ABA concentration
gradient and the transpiration stream. The relationships
between stomatal conductance and fluorescence parameters
depended on the actinic light intensity, i.e. NPQ was greaterand FPSII was lower at high light intensity. The fluorescence parameters did not change, regardless of stomatalclosure levels at a photosynthetically active photon flux
(PPF) of 270 mmol m–2 s–1; however, they drasticallychanged at PPF values of 350 and 700 mmol m–2 s–1, when
the total stomatal conductance decreased to less than 80
and 200 mmol m–2 s–1, respectively. This study has, for the
first time, quantitatively analyzed relationships between
spatiotemporal variations in stomatal conductance and fluorescence parameters in intact leaves under aerobic conditions.
Keywords: Chlorophyll fluorescence imaging — Nonphotochemical quenching — Phaseolus vulgaris L. — Photochemical yield of photosystem II — Stomatal conductance —Thermal imaging.
Abbreviations: ABA, abscisic acid; ap, absorption coefficient ofshort-wavelength radiation by the leaf; DH, thermal diffusivity in air;
DW, water vapor diffusivity in air; e, emissivity of long-wavelength
radiation of the leaf; Es, short-wavelength radiation from the environment; Ew, long-wavelength radiation from the environment; iF, fluorescence intensity image measured under actinic light; iFm, fluorescenceintensity image measured during a saturation light pulse during darkness after exposure of the leaf to darkness for at least 1 h; iFm¢, fluorescence intensity image measured during a saturation light pulse during
steady-state photosynthesis; gaH, boundary layer conductance to heattransfer; gaW, boundary layer conductance to water vapor diffusion; gsl,
stomatal conductance to water vapor diffusion of the lower leaf sur1;
face; gst, total stomatal conductance to water vapor diffusion; gsu,
stomatal conductance to water vapor diffusion of the upper leaf
surface; h, relative humidity of air; L, latent heat of evaporation; NPQ,
non-photochemical quenching; PAR, photosynthetically active radiation; FPSII, photochemical yield of photosystem II; PPF, photosynthetically active photon flux; PSI, photosystem I; PSII, photosystem II; rcp,
volumetric heat capacity of air; s, Stefan–Boltzmann constant; Ta, airtemperature; Tl, leaf temperature; W, transpiration rate.

Introduction
Progress in imaging techniques now permits us to visualize spatiotemporal variations in invisible plant responses to various stresses, which cannot be detected by conventional point
data measurements (e.g. Ellenson and Amundson 1982, Omasa
et al. 1985, Omasa and Aiga 1987, Hashimoto et al. 1990,
Omasa 1990, Kramer and Boyer 1995, Lichtenthaler et al.
1996, Chaerle and Straeten 2000, Govindjee and Nedbal 2000,
Häder 2000, Omasa et al. 2002). In particular, at the level of an
intact leaf, thermal imaging and chlorophyll fluorescence imaging techniques have been widely used to assess the dynamics
and the heterogeneity of stomatal responses, called stomatal
patchiness (Terashima et al. 1988, Terashima 1992, Beyschlag
and Eckstein 1998), and the photosynthetic activity of the leaf
surface.
Thermal imaging has often been used to remotely measure dynamic, heterogeneous distributions of leaf temperature,
as a surrogate for direct measures of stomatal conductance(Horler et al. 1980, Omasa et al. 1980, Hashimoto et al. 1984,
Omasa and Aiga 1987, Raskin and Ladyman 1988, Jones
1999). Stomatal conductance is an indicator of the extent of
stomatal opening and indirectly indicates the rate of photosynthetic activity, i.e. CO2 assimilation (Jones 1992). Recent studies have indicated the importance of quantitative evaluation of
stomatal conductance from the measured leaf temperature andthe basic energy-balance equation (Inoue et al. 1990, Taconet etal. 1995, Jones 1999). We previously reported that measuring
the leaf temperature under strictly controlled environmental
conditions allowed straightforward, quantitative evaluation of
stomatal conductance to water vapor diffusion across the leaf
surface (Omasa et al. 1981a, Omasa and Croxdale 1992). At

Corresponding author: E-mail, aomasa@mail.ecc.u-tokyo.ac.jp; Fax, +81-3-5841-8175.

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Imaging of stomatal conductance, NPQ, and FPSII

Fig. 1 Visual image of a measurement area of an attached P. vulgaris L. leaf used for the experiment performed at a PPF of 350 mmol
m–2 s–1. The white area represents an ABA-treated region, as determined by a superimposed thermal image obtained immediately after
the ABA treatment. Symbols: the circle (site 1) is a mesophyll site
separated by the midvein from the ABA-treated region, the triangle(site 2) represents the mesophyll site at the center of the ABA-treatedregion, and the square (site 3) represents a mesophyll site 6–7 mm
away from the ABA-treated region along a lateral vein toward the leaf
margin. Line X–X¢ shows the profile analysis line used in the following analyses.

that time, this approach was applied only to an analysis of theeffects of air pollutants on stomatal conductance (Omasa et al.
1981b, Omasa et al. 1981c).
Chlorophyll fluorescence results from absorbed lightenergy that was not used for photosynthetic reactions and heatdissipation. Chlorophyll fluorescence imaging provides information on photosynthetic activities without destruction of orcontact with the living leaf (Omasa et al. 1987, Daley et al.
1989, Croxdale and Omasa 1990, Daley 1995, Rolfe andScholes 1995, Lichtenthaler and Miehé 1997, Govindjee and
Nedbal 2000, Omasa et al. 2002). Many useful fluorescenceparameters, such as NPQ (non-photochemical quenching) andFPSII (photochemical yield of photosystem II (PSII)), have beendeveloped and used as proxies of photosynthetic activity underactinic light (Genty et al. 1989, Krause and Weis 1991,
Govindjee 1995, Maxwell and Johnson 2000, Müller et al.
2001). An image of NPQ indicates the distribution and thestrength of the intrathylakoid pH gradient and the ability ofchloroplasts to dissipate excess excitation energy as heat on theleaf (Daley et al. 1989, Osmond et al. 1998, Müller et al. 2001).
Therefore, NPQ images have been used as indicators of stomatal patchiness, because heat dissipation depends on stomatalclosure (Daley et al. 1989, Mott 1995, Eckstein et al. 1996,
Osmond et al. 1998). FPSII images indicate the distribution of
the yield of linear electron transport through PSII (Genty andMeyer 1995, Bro et al. 1996, Meyer and Genty 1998, Meyerand Genty 1999). Under anaerobic conditions, a FPSII image
can be used as a relative map of CO2 assimilation and stomatal

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patchiness, because photorespiration and the Mehler reaction
are completely inhibited under such conditions (Genty andMeyer 1995, Siebke and Weis 1995). Meyer and Genty (1998)
demonstrated that quantitative calculation of stomatal conductance to water vapor diffusion on a leaf surface could beachieved with a FPSII image measured under anaerobic conditions. However, the effects of anaerobic conditions on stomatal
conductance and other photosynthetic activities are controversial (Osmond 1981, Ort and Baker 2002).
In this study, we therefore developed a new imaging system capable of concurrent, quantitative, straightforward evaluation of stomatal conductance, NPQ, and FPSII in intact, attachedleaves solely under aerobic conditions, by using both thermal
imaging and chlorophyll fluorescence imaging. With this system, we quantitatively investigated relationships between spatiotemporal variations of stomatal conductance, NPQ, and FPSII
across intact Phaseolus vulgaris L. leaves in response to abscisic acid (ABA) treatment at individual small sites under threeintensities of actinic light.

Results
Fig. 1 shows a visual image of P. vulgaris L. leaf within a
2.6´2.6 cm leaf area in the center of the 5´5 cm measurement
window (see Materials and Methods) used for the experiment
at a photosynthetically active photon flux (PPF) of 350 mmol
m–2 s–1. The white area represents an ABA-treated region, as
determined by thermal imaging immediately after the ABA
treatment.
Fig. 2 shows changes in the distribution of total stomatal
conductance values within the same area as in Fig. 1 from 0 to
90 min after ABA treatment under illumination at a PPF of
350 mmol m–2 s–1. The stomatal conductance values along line
X–X¢ (shown in Fig. 1) are represented below each image.
Before ABA treatment, stomatal conductance was uniformly
distributed in the mesophyll areas, with values ranging from
449 to 512 mmol m–2 s–1, except in the immediate vicinities ofthe mid and lateral veins (Fig. 2A). Immediately after ABA
treatment, stomatal conductance decreased within and aroundthe ABA-treated region, and the area of decreased conductancespread laterally with time. The decrease in stomatal conductance at the center of the treated region (i.e. site 2 in Fig. 1) was
especially strong. The conductance was 193 mmol m–2 s–1 after
15 min, 125 mmol m–2 s–1 after 30 min, and only 56 mmol
m–2 s–1 after 50 min, after which the value stayed in the range
56 to 64 mmol m–2 s–1 (Fig. 3). The area of decreasing stomatal
conductance expanded in all directions for 50 min after ABA
treatment (Fig. 2B, C); subsequently, it spread mainly along thelateral vein toward the leaf edge between 50 and 90 min after
treatment (Fig. 2C, D). At an interveinal mesophyll site (site 3
in Fig. 1) that was 6–7 mm away from the ABA-treated region
toward the leaf edge along a lateral vein, stomatal conductanceshowed little change during the initial 30 min after treatment,
but it gradually decreased to 305 mmol m–2 s–1 at 60 min and1292

Imaging of stomatal conductance, NPQ, and FPSII

Fig. 2 Changes in the distribution of total stomatal conductance values (indicated by color) in the measurement area shown in Fig. 1 from 0 to
90 min after ABA treatment. The cross-sectional diagrams represent transects of total stomatal conductance values across the total stomatal conductance images along the line X–X¢ shown in Fig. 1. The region within the dotted line (B–D) is the ABA-treated region. Experimental conditions
were as follows: air temperature, 26.5°C; relative humidity, 48%; short-wavelength radiation, 44.5 W m–2; long-wavelength radiation, 0.98 kW m–2;
actinic light intensity, PPF = 350 mmol m–2 s–1; and boundary-layer conductance for heat transfer, 12.0 mm s–1. (A) Just before ABA treatment;(B) 15 min after treatment; (C) 50 min after treatment; (D) 90 min after treatment.

Fig. 3 Time courses of total stomatal conductance from 0 to 90 min after ABA treatment at
each site indicated in Fig. 1. Symbols: circles (site 1), triangles (site 2), squares (site 3).

Fig. 4 Changes in the distribution of NPQ (indicated by color) in the measurement area shown in Fig. 1 from 0 to 90 min after ABA treatment.
The cross-sectional diagrams represent transects of NPQ values across the NPQ images along the line X–X¢ shown in Fig. 1. The region within
the dotted line (B–D) is the ABA-treated region. Experimental conditions were as in Fig. 2. (A) Just before ABA treatment, and (B) 15 min, (C)50 min, and (D) 90 min after ABA treatment.

Imaging of stomatal conductance, NPQ, and FPSII

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Fig. 5 Changes in the distribution of FPSII (indicated by color) in the measurement area shown in Fig. 1 from 0 to 90 min after ABA treatment.
The cross-sectional diagrams represent transects of FPSII values across the FPSII images along the line X–X¢ shown in Fig. 1. The region within thedotted line (B–D) is the ABA-treated region. Experimental conditions were as in Fig. 2. (A) Just before ABA treatment, and (B) 15 min, (C)50 min, and (D) 90 min after ABA treatment.

215 mmol m–2 s–1 at 90 min (Fig. 3). The stomatal conductanceat an interveinal mesophyll site (site 1 in Fig. 1) separated by
the midvein from the ABA-treated region showed no change
and stayed between 457 and 475 mmol m–2 s–1 throughout theexperiment (Fig. 3).
Fig. 4 and 5 show changes in the distributions of NPQ andFPSII in the same leaf area as in Fig. 1 and 2 from 0 to 90 min
after the ABA treatment. The NPQ and FPSII values along line
X–X¢ (shown in Fig. 1) are represented below each image.
Before ABA treatment, the NPQ and FPSII values were uni-

formly distributed across the leaf area, with values in theranges of 0.16 to 0.62 for NPQ and 0.72 to 0.77 for FPSII
except at the veins. Until 30 min after the ABA treatment, nochange in NPQ or FPSII was seen in the images or the timecourses (Fig. 4–6). Thereafter, NPQ began to increase at thesites of severe stomatal closure and continued to increase withtime (Fig. 6A). The most affected area were the same in Fig. 2
and 4. The NPQ value at site 2 reached 1.19 at 50 min and 1.50
at 90 min after the treatment (Fig. 6A). Increased NPQ values
were limited to the areas showing severe stomatal closure, andFig. 6 Time courses of NPQ (A) and FPSII (B) values
from 0 to 90 min after ABA treatment at each site indicated in Fig. 1. Symbols: circles (site 1), triangles (site 2),
squares (site 3).

Fig. 7 Relationships between total stomatal conductance and fluorescence parameters, NPQ (A) and FPSII(B), from 0 to 90 min after ABA treatment. Symbols:
blue circles, green squares, yellow diamonds, and red
triangles represent 0, 15, 50, and 90 min after ABA
treatment, respectively. The 120 points plotted for eachmeasurement time were taken along the line X–X¢
shown in Fig. 1.

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Imaging of stomatal conductance, NPQ, and FPSII

Fig. 8 Effects of ABA treatment on
the distribution of total stomatal conductance, NPQ, and FPSII on P. vulgaris L. leaves 90 min after ABA
treatment under illumination at PPFs
of 270 (A) and 700 (B) mmol m–2 s–1.
Other experimental conditions wereas follows for PPFs of 270 and
700 mmol m–2 s–1, respectively: airtemperature, 26.5 and 25.3°C; relative humidity, 45% and 53%; shortwavelength radiation, 34.3 and88.9 W m–2; long-wavelength radiation, 0.98 and 1.16 kW m–2; andboundary layer conductance for heattransfer, 14.5 and 34.4 mm s–1.

Fig. 9 Relationships between total stomatal conductance and fluorescence parameters, NPQ (A) and FPSII(B), 90 min after ABA treatment under illumination at
PPFs of 270 (blue circles), 350 (red triangles), and 700(yellow diamonds) mmol m–2 s–1. These data wereobtained at sites along the lines X–X¢, Y–Y¢, Z–Z¢
shown in Fig. 1 and 8 and correspond to the data shownin Fig. 2D, 4D, 5D, 8A, and 8B.

slight patchiness is also evident in these areas (Fig. 4C, D). The
NPQ at sites 1 and 3 showed little change with time andremained in a range of 0.35 to 0.53 for 90 min, with a slightincreasing trend; however, the increase was very small compared with the increase at site 2 induced by ABA treatment(Fig. 6A). FPSII slightly decreased, but only in the areas thatshowed the most severe stomatal closure; the decrease at site 2
became evident only after 30 min, reaching 0.67 at 50 min after

treatment (Fig. 6B). Afterwards, FPSII stayed in the range of
0.65 to 0.68. Slight patchiness was observed in those sites (Fig.
5C, D). FPSII at sites 1 and 3 showed no detectable change andremained in the range of 0.73 to 0.74 for 90 min (Fig. 6B).
Fig. 7 shows relationships between the total stomatal conductance and NPQ and FPSII at sites along the line X–X¢ shownin Fig. 1. NPQ stayed in the range of 0.16 to 0.69 when the
total stomatal conductance was greater than 80 mmol m–2 s–1;

Imaging of stomatal conductance, NPQ, and FPSII

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Fig. 10 Schematic diagram of the thermaland chlorophyll fluorescence imaging system. Image measurements were performed
in an environmentally controlled measuring
chamber. Environmental conditions (air temperature, relative humidity, short-wavelength
radiation, long-wavelength radiation, and air
current around the leaf surfaces) were kept
uniform and constant during the experiment. An attached leaf was placed in an
opaque cardboard frame (15´20 cm) with a
5´5 cm window and set horizontally in themeasuring chamber.

however, it drastically increased as the total stomatal conductance values fell below 80 mmol m–2 s–1. Before ABA treatment,
most of the low-NPQ points (blue closed circle in Fig. 7A)
with total stomatal conductance values greater than 450 mmol
m–2 s–1 were located in mesophyll areas. The low-NPQ points
moved towards the left and became the upper points with timeafter ABA treatment. Meanwhile, changes in FPSII in relation tototal stomatal conductance were very small; however, the pattern was somewhat similar to that of NPQ. The FPSII values
stayed in the range 0.69 to 0.77 when the total stomatal conductance was greater than 80 mmol m–2 s–1 and decreased
somewhat when the total stomatal conductance was less than
80 mmol m–2 s–1.
Fig. 8 shows the distributions of total stomatal conductance, NPQ, and FPSII on P. vulgaris L. leaves 90 min after ABA
treatment under illumination at PPFs of 270 (Fig. 8A) and
700 mmol m–2 s–1 (Fig. 8B). At the lower photon flux, the areas
within and around the ABA-treated region showed a significant decrease in total stomatal conductance, which was
72 mmol m–2 s–1 at the center of the ABA-treated region. However, the NPQ and FPSII values in these areas did not change.
The values of total stomatal conductance, NPQ, and FPSII in theareas not affected by the ABA treatment stayed in the ranges of
336 to 381 mmol m–2 s–1, 0.26 to 0.47, and 0.76 to 0.82, respectively (Fig. 8A). At the higher photon flux, the areas within andaround the ABA-treated region showed a decrease in total stomatal conductance, a strong increase in NPQ, and a marked
decrease in FPSII. At the center of the ABA-treated region, thetotal stomatal conductance, NPQ, and FPSII were 125 mmol m–2
s–1, 4.37, and 0.18, respectively (Fig. 8B). The values of total
stomatal conductance, NPQ, and FPSII in the areas not affectedby the ABA treatment were in the ranges 370 to 430 mmol m–2
s–1, 1.67 to 2.31, and 0.37 to 0.45, respectively (Fig. 8B).
Fig. 9 shows relationships between the total stomatal conductance and NPQ and FPSII 90 min after ABA treatment underillumination at PPFs of 270 (blue closed circle), 350 (redclosed triangle), and 700 (yellow diamond) mmol m–2 s–1. At
the lowest photon flux, NPQ and FPSII did not change, regardless of the decrease in the total stomatal conductance. At theintermediate level of photon flux, the fluorescence parameters
remained constant at total stomatal conductance values greaterthan 80 mmol m–2 s–1, but they changed drastically below
80 mmol m–2 s–1, as mentioned above (see Fig. 7). At the highest photon flux, the NPQ and FPSII values stayed in the ranges
1.67 to 2.67 and 0.33 to 0.45, respectively, at total stomatal
conductance values greater than 200 mmol m–2 s–1. However,the fluorescence parameters changed drastically when the total
stomatal conductance decreased to less than about 200 mmol
m–2 s–1. The general patterns of the relationships between the
total stomatal conductance and the fluorescence parameters
were similar to those observed at the intermediate PPF, eventhough the absolute values were very different.

Discussion
A new imaging system capable of concurrent, quantitative,
straightforward evaluation of stomatal conductance, NPQ, andFPSII of intact attached leaves solely under aerobic conditions,using both thermal imaging and chlorophyll fluorescence imaging was developed. Using this system, we investigated relationships between the spatiotemporal variations of stomatal
conductance and of NPQ and FPSII across intact P. vulgaris L.

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Imaging of stomatal conductance, NPQ, and FPSII

leaves in response to ABA treatment at each 0.1´0.1 mm site
under three actinic light intensities.
Stomatal conductance is an indicator of the extent of stomatal opening: it decreases when the stomata close and viceversa (Omasa et al. 1983, Jones 1992). Spatiotemporal variations in the total stomatal conductance were evaluated fromleaf temperature images measured by thermal imaging underconstant thermal conditions. In experiments performed at PPFs
of 270, 350, and 700 mmol m–2 s–1, the distributions of total stomatal conductance in the mesophyll areas before ABA treatment were constant (e.g. Fig. 2A). The mean value of the total
stomatal conductance ranged from 330 to 512 mmol m–2 s–1,
which indicates that the stomata were well opened (Omasa etal. 1983, Omasa and Croxdale 1992, Jones 1999). In an experiment under illumination at a PPF of 350 mmol m–2 s–1, the total
stomatal conductance in the ABA-treated region rapidly
decreased in the first 15 min after the ABA treatment (Fig. 2B).
In particular, the center of the ABA-treated region (Fig. 3)
showed a substantial decrease in total stomatal conductance(approximately an 80% decrease) compared with the rest of the
ABA-treated region. The total stomatal conductance continued
to decrease to less than 60 mmol m–2 s–1 by 50 min after the
ABA treatment (Fig. 2C, 3). Thereafter, it stopped and stayedat the minimum value of 60 mmol m–2 s–1 until 90 min after the
ABA treatment (Fig. 2D, 3). These results show that ABA
induced rapid stomatal closure and diminished the total stomatal conductance to a minimum value in less than an hour.
The minimum value of the total stomatal conductance did notreach zero, however. One reason may be that the ABA treatment could not induce complete stomatal closure. Another may
be that cuticular transpiration was not negligible when the stomatal apertures were severely closed (Meyer and Genty 1998).
Or third, the calculation of stomatal conductance from leaf
temperature may have had an error (Omasa et al. 1981a, Omasa
and Croxdale 1992).
Spatiotemporal variations in the total stomatal conductance revealed the diffusion of ABA in the leaf tissue (Fig. 2).
Although the area of low total stomatal conductance expanded
in almost all directions from 15 to 50 min after the ABA treatment (Fig. 2B, C), the rate at which the area with low total stomatal conductance expanded toward the leaf margin along alateral vein was faster than in other directions between 50 and
90 min after ABA treatment (Fig. 2C, D). These results showthat the initial expansion of the area with low total stomatal
conductance depended on ABA diffusion, induced by the ABAconcentration gradient through apoplasts and veinlets, and thesubsequent expansion toward the leaf edge depended mainly on
ABA transportation by the transpiration stream in the vein, in
addition to the former mechanism of expansion. This interpretation is confirmed by the time course of changes in total
stomatal conductance at a site 6–7 mm away from the ABAtreated region, closer to the leaf margin along a lateral vein(site 3 in Fig. 1 and 3). The initial rate of expansion wasapproximately 11.4 mm h–1, and this rate was maintained along

the lateral vein (Fig. 2D). On the other hand, total stomatal
conductance at a mesophyll site separated by the midvein fromthe ABA-treated region remained at the initial values throughout the experiment, indicating that ABA did not cross the midvein (see site 1 in Fig. 1–3).
At PPFs of 270 and 700 mmol m–2 s–1, the values of total
stomatal conductance at the sites not affected by ABA treatment were relatively lower than at a PPF of 350 mmol m–2 s–1(Fig. 2D, 8, 9). This may be attributed to differences in theindividual leaves used for the experiments and in the environmental conditions, including the light intensities. At 90 min
after ABA treatment, the minimum value, approximately
125 mmol m–2 s–1, of total stomatal conductance occurred at a
PPF of 700 mmol m–2 s–1; this value was higher than thoseobtained at PPFs of 270 and 350 mmol m–2 s–1 (Fig. 9). Thismay have been due to differences in the amount of ABA solution absorbed by the leaves.
Chlorophyll fluorescence imaging reveals the effects of
ABA treatment on non-photochemical quenching and on theyield of linear electron transport through PSII. At a PPF of
350 mmol m–2 s–1, NPQ and FPSII values were uniformly distributed over the leaf surface before ABA treatment (Fig. 4A,
5A). The mean value of NPQ was low and that of FPSII was
high, because the experiment was performed under aerobic
conditions (Bro et al. 1996, Meyer and Genty 1998, Meyer andGenty 1999). But 15 min and even 90 min after ABA treatment, mesophyll areas with total stomatal conductance values
greater than about 80 mmol m–2 s–1 and without any change in
NPQ or FPSII were observed (Fig. 2, 4, 5). This shows that
ABA treatment induced only stomatal closure, without affecting the non-photochemical quenching or the yield of linear
electron transport through PSII in these mesophyll areas(Fig. 7). The lack of changes in FPSII suggested that photorespiration was active and consumed any excess light energy inthose areas, thus protecting the photosynthetic apparatus fromphotoinhibitory damage (Osmond 1981, Sharkey et al. 1988,
Cornic and Briantais 1991, Brestic et al. 1995, Ort 2001, Ort
and Baker 2002). Consequently, the NPQ in those areas did notchange, because an extreme intrathylakoid pH gradient was not
generated, owing to cooperative consumption of light energy
by CO2 fixation and photorespiration (Müller et al. 2001). Thisphenomenon was also observed at a PPF of 270 mmol m–2 s–1,
even though the total stomatal conductance reached a minimum value of approximately 70 mmol m–2 s–1 (blue closed circles in Fig. 9). Moreover, a similar phenomenon occurred at a
PPF of 700 mmol m–2 s–1 when the total stomatal conductancewas over 200 mmol m–2 s–1 (yellow closed diamonds in Fig. 9).
In contrast, in the experiment at a PPF of 350 mmol m–2 s–1,
when stomatal apertures were severely closed and the total stomatal conductance was extremely decreased to less than
80 mmol m–2 s–1 at 50 min after ABA treatment, FPSII began todecrease slightly at the center of the ABA-treated region (Fig.
2C, 5C, 6B, 7B). Simultaneously, NPQ began to increase in
those areas (Fig. 2C, 4C, 6A, 7A). This suggests that, when theImaging of stomatal conductance, NPQ, and FPSII

Fig. 11 A comparison of gaH calculated from Eq. (2) and gaH calculated from Eq. (7) under several air current conditions at a PPF of
350 mmol m–2 s–1. Long-wavelength radiation from the environment(Ew) was evaluated by substituting gaH in Eq. (2). The absorption coefficient of the short-wavelength radiation of the black felt model leaf
was 0.98±0.02. The long-wavelength emissivity of the model leaf
was 0.98±0.02. The experimental conditions were as follows: shortwavelength radiation, 44.5 W m–2; air temperature, 26.5±0.1°C; andrelative humidity, 48%.

stomatal apertures were severely closed and the CO2 supply
through the stomata was severely inhibited, the linear electrontransport chain became over-reduced, and cyclic electron transport around PSI and electron transport to the water–water cycle
were activated (Schreiber and Neubauer 1990, Asada 1999, Ort
2001, Ort and Baker 2002). These alternative electron transport systems generate a large intrathylakoid pH gradient andactivate dissipation of the excess light energy as heat by meansof the xanthophyll cycle (Müller et al. 2001, Ort 2001, Ort and
Baker 2002). Our results showed that, when a slight decrease in
the yield of linear electron transport through PSII (FPSII) was
observed, heat dissipation of the excess light energy was activated, to protect the photosynthetic apparatus from photoinhibitory damage. This phenomenon was also observed at a
PPF of 700 mmol m–2 s–1 when the total stomatal conductancedecreased to less than 200 mmol m–2 s–1 (yellow closed diamonds in Fig. 9). However, this phenomenon was not apparent
in the experiment under illumination at a PPF of 270 mmol m–2
s–1 (blue closed circles in Fig. 9), because the light intensity
was too weak to activate the heat-dissipation process.
We interpret the relationships between the total stomatal
conductance and the fluorescence parameters at the various
actinic light intensities as follows. If the actinic light intensity
is weak enough (e.g. PPF = 270 mmol m–2 s–1 in our experiment), the fluorescence parameters do not change even though
the total stomatal conductance reaches its minimum value. If
the actinic light intensity is strong enough (e.g. PPF = 350 or
700 mmol m–2 s–1 in our experiment), the fluorescence parameters change drastically when the total stomatal conductancedecreases to less than a specific value. The specific value ishigher at higher actinic light intensities (e.g. 80 mmol m–2 s–1

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for 350 mmol m–2 s–1 and 200 mmol m–2 s–1 for 700 mmol m–2 s–1).
Such specific values cannot be detected by conventional point
data measurements, because the conventional point data measurements measure average values of a selected leaf area thatshows heterogeneous responses to environmental stimuli(Omasa 1990, Omasa and Croxdale 1992, Terashima 1992,
Beyschlag and Eckstein 1998). For example, it has beenreported that the heat-dissipation process is gradually activatedwith respect to decreasing stomatal conductance during water
stress (Flexas et al. 2002). However, in our experiment, image
analysis of the relationship between stomatal conductance andchlorophyll fluorescence parameters at a resolution of 0.1 mm
revealed that the heat-dissipation process is rapidly activatedonly when the absorbed light energy exceeds energy consumption by CO2 fixation and photorespiration within each mesophyll site. Our study has, for the first time, quantitatively analyzed relationships between spatiotemporal variations in total
stomatal conductance and chlorophyll fluorescence parameters
on a 0.1´0.1 mm grid of intact leaves under exclusively aerobic conditions, by using both thermal imaging and chlorophyll
fluorescence imaging.

Materials and Methods
Plant material
Kidney bean (P. vulgaris L. cv. ‘Shin-edogawa’) seeds weresown in pots (12 cm in diameter and 10 cm tall) in an environmentallycontrolled growth chamber, and the plants were grown for 4 weeks.
The pots were filled with artificial soil (mixture of vermiculite and perlite, 1 : 1, v/v). The plants were illuminated for 12 h each day with fluorescent lights at a PPF of 300 mmol m–2 s–1. Air temperature was
26.5°C during the day and 20.0°C at night. Relative humidity was 45%
during the day and 60% at night. Plants were watered daily with anutrient solution (1 : 1,000 dilution of HYPONeX). Fully expanded
mature leaves were used in situ for the experiments. The ratio of thestomatal frequency on the upper surface to that on the lower surface
was about 1 : 8.2. The leaves were 0.08 to 0.15 mm thick. The difference in leaf temperature between upper and lower surfaces was about
0.1°C at a PPF of 350 mmol m–2 s–1.
Computer-aided imaging system for combining thermal imaging andchlorophyll fluorescence imaging
Fig. 10 shows a scheme of the computer-aided imaging systemfor combining thermal imaging and chlorophyll fluorescence imaging.
Thermal images, for calculating stomatal conductance, were measured
with an optical-mechanical scanning thermographic system (JEOL,
JTG-5200, thermal camera and controller) with an HgCdTe detector(8–13 mm, cooled by liquid nitrogen) having a temperature resolutionof 0.05°C. The detected signals were converted into 16-bit resolution
digital signals (512 horizontal ´ 480 vertical pixels) and recorded asdigital data to a hard disk in the thermal camera controller. After thedata were transferred to the computer, they were converted to exacttemperature images by using an equation determined by comparing thetemperatures of dry and wet model leaves with black felt surfaces onboth sides (the absorption coefficient of short-wavelength radiation
was 0.98 and the emissivity of long-wavelength radiation was 0.98 in
the model leaf) as measured with the thermographic system and withthermocouples (Omasa et al. 1981a, Omasa and Croxdale 1992). The
distance from the thermal camera to the leaf surface was 0.35 m, andthe spatial resolution of the thermal image was about 0.1 mm. By
measuring the thermal image of leaf having a drastic change in the1298

Imaging of stomatal conductance, NPQ, and FPSII

temperature distribution, we confirmed that the effect of heat conduction on the spatial distribution of leaf temperature was small.
Chlorophyll fluorescence images were measured with a cooledcharge-coupled device (CCD) video camera (Hamamatsu Photonics,
C5985-02) equipped with an interference filter (Optical Coatings
Japan, IF-W, l = 683 nm, half-band width = 10 nm) and a long-passfilter (Corning, 2–64; l >640 nm). The images were recorded on a digital video recorder (Sony, DSR-V10) at 640 horizontal ´ 480 vertical
pixels per frame with 8-bit resolution and were analyzed by selfproduced software. Continuous actinic light (PPF values of 270, 350,
and 700 mmol m–2 s–1) for photosynthesis was provided by a 150-W
halogen lamp (Nikon, PSM-11520) equipped with a short-pass filter(Corning, 4–96; l <620 nm) and was passed through two heatabsorbnsing filters via an optical fiber system. A saturation light pulse(PPF 2,800 mmol m–2 s–1 for 1 s), to cause a transient saturation of photochemistry, was provided by two 180-W metal halide lamps (Sumita
Optical Glass, Inc., LS-M180) and passed through a short-pass filter(Corning, 4–96; l <620 nm) and a heat-absorbing filter via an opticalfiber system.
An in-situ leaf (i.e. a leaf that remained attached to the plant) was
sandwiched between two opaque pieces of cardboard (15´20 cm) having a 5´5 cm window in the same place. The sandwich was mounted
horizontally in the measuring chamber containing an atmosphere of
approximately 21% O2 and 560 ppm CO2 (Fig. 10). The spatial distributions of the boundary-layer conductance of the upper and lower surfaces of the leaf within the window were kept constant by controllingthe air current with small electric fans. The actinic light intensity andother environmental conditions such as air temperature, relative
humidity, short-wavelength radiation, and long-wavelength radiation
were also kept constant over the leaf area in the window. Thermal
imaging and chlorophyll fluorescence imaging were performed simultaneously after the leaf’s photosynthesis reached steady state. The
measured images were analyzed by both self-produced software andcommercial software (ERDAS, ERDAS IMAGINE Ver. 8.3). Geometrical deformation of images was corrected by affine transformation
within an error of 1 pixel at each position regardless of image type.
Calculation of total stomatal conductance image from leaf temperature image
Spatial distributions of stomatal conductance to water vapor diffusion across a leaf surface can be quantitatively evaluated from theleaf temperature image as measured by a thermographic system(Omasa et al. 1981a, Omasa and Croxdale 1992). In this study, thetotal stomatal conductance was computed as the sum of the values onboth sides of the leaf, although the stomatal conductance of the uppersurface differed from that of the lower surface (Omasa et al. 1983,
Jones 1992).
Water evaporates from mesophyll and epidermal cell walls anddiffuses into the air through the stomata and boundary layer. If the leaf
temperature and boundary layer are assumed to be equal on both sides,
the transpiration rate, Wx (g mm–2 s–1), at each leaf site is expressed by
X ( T lx ) – hX ( T a ) X ( T lx ) – hX ( T a )W x = ----------------------------------------- + ----------------------------------------1 ¤ g sux + 1 ¤ g aWx 1 ¤ g slx + 1 ¤ g aWx(1)

where Tlx is the leaf temperature (°C), Ta is the air temperature (°C),
X(T) is the saturated water vapor density at T °C (g mm–3), h is the relative humidity of the air, gsux is the stomatal conductance to water
vapor diffusion of the upper leaf surface (mm s–1), gslx is the stomatal
conductance to water vapor diffusion of the lower leaf surface (mm s–1),
and gaWx is the boundary layer conductance to water vapor diffusion(mm s–1). The subscript x denotes the values at the local site (x) on theleaf. Because the difference in leaf temperature between the upper andlower surfaces was about 0.1°C, this assumption was reasonable.
Considering the heat balance at each leaf site x, the transpiration
rate at the leaf site can be described by the following simplified equation:

4
1
W x = ------ ´ [ a p E sx + e { E wx – 2s ( 273.15 + T lx ) }L+ 2g aHx rc p ( T a – T lx )](2)where Esx is the short-wavelength radiation from the environment (Wm–2 at wavelengths £3 mm), Ewx is the long-wavelength radiation fromthe environment (W m–2 at wavelengths ³3 mm), ap is the absorption
coefficient of short-wavelength radiation of the leaf, e is the emissivityof long-wavelength radiation of the leaf, s is the Stefan–Boltzmann
constant (5.67´10–8 W m–2 K–4), gaHx is the boundary layer conductance to heat transfer (mm s–1), rcp is the volumetric heat capacity of air(1.192 kW s m–3 °C–1), and L is the latent heat of evaporation (2.44 kW
s g–1). When the environmental conditions such as air temperature, relative humidity, radiation (long-wavelength and short-wavelength), andair current (boundary layer conductance) around the leaf surface are
kept constant at the leaf site, only the leaf temperature, Tlx, remains as
a variable on the right side of Eq. (2). Therefore, the transpiration rate
at the leaf site can be evaluated from the measured leaf temperature (Tlx).
Meanwhile, gaWx used in Eq. (1) can be expressed with gaHx usedin Eq. (2):gaWx = (DW / DH)2/3gaHx(3)where DH is the thermal diffusivity in air (22.3 mm2 s–1), and DW is thewater vapor diffusivity in air (25.1 mm2 s–1) (Jones 1992). Consequently, the total stomatal conductance to water vapor diffusion at eachleaf site can be evaluated from Eqs. (1), (2), and (3) when the parameters other than Tlx are known. To be specific, Wx is first calculated by
Eq. (2) from the leaf temperature (Tlx). Second, Wx in Eq. (1) is substituted for the value of Wx determined from Eq. (2). At the same time,
gaWx in Eq. (1) is substituted for gaHx/(DH/DW)2/3 determined from Eq.
(3), and gsux in Eq. (1) is substituted for gslx multiplied by the ratio of
the number of stomata on the upper surface to those on the lower surface. Finally, gslx in Eq. (1) is determined by solving the quadratic
equation; subsequently, the total stomatal conductance to water vapor
diffusion (gstx) is computed as the sum of the values from each side(heat balance method):
g stx = g sux + g slx
R st + 1ö
= æ --------------- gè R st ø slx(4)

where Rst is the ratio of the number of stomata on the lower leaf surface to those on the upper leaf surface. The units of total stomatal conductance to water vapor diffusion were converted into molar units(mmol m–2 s–1) from mass units (mm s–1) (40 mmol m–2 s–1 = 1 mm s–1;
Jones 1999).
Calculation of NPQ and FPSII images from a set of fluorescence intensity images
A set of fluorescence intensity images (iF, iFm¢, and iFm) wasused for the calculation of the NPQ and FPSII images. After stomatal
opening reached steady state under a given light flux, an image iF was
measured. Just after the measurement, an iFm¢ image was measured
during a saturation light pulse during steady-state photosynthesis.
Finally, an iFm image was measured during a saturation light pulse
after the leaf was held in darkness for at least 1 h after the experiments.
Each fluorescence intensity image (iF, iFm¢, and iFm) was divided by
the average value of fluorescence emitted from a fluorescent standard
induced by the corresponding light intensity to convert it into a relative fluorescence yield image.
Using the relative fluorescence yield images, images (640 horizontal ´ 480 vertical pixels, 8-bit resolution) of NPQ and FPSII weremade by computing each pixel with the following equations (Genty etal. 1989, Bilger and Björkman 1990, Maxwell and Johnson 2000):

Imaging of stomatal conductance, NPQ, and FPSII
i

i

i

i

Fm ¤ R SL –( Fm¢) ¤ R SL
Fm – Fm ¢NPQ = ---------------------------------------------------- = ----------------------(5)iiFm ¢ ¤ R SL
Fm ¢
( )
and
( )
i
i
( Fm¢) ¤ R SL – F ¤ R AL
(6)
F PSII = ---------------------------------------------------iFm ¢ ¤ R SL
( )where RSL and RAL are the average values of fluorescence emitted fromthe fluorescent standard induced by the saturation light pulse and theactinic light, respectively.
The NPQ image, with pixel values ranging from zero to infinity,
represents the extent of the intrathylakoid pH gradient and the abilityof chloroplasts to dissipate excess excitation energy as heat (Bilger and
Björkman 1990, Maxwell and Johnson 2000). The FPSII image represents the yield of linear electron transport through PSII, with pixel values ranging from 0 to 1 (Genty et al. 1989, Maxwell and Johnson
2000).
Measurement of thermal environments and parameters
To calculate the total stomatal conductance by the abovementioned heat-balance method, the thermal environment and theparameters in Eq. (1) and Eq. (2) have to be known. The air temperature (Ta) and relative humidity (h) in the measuring chamber were kept
at 26.5±0.1°C and 48% for PPFs of 270 and 350 mmol m–2 s–1 and at
25.3±0.1°C and 53% for the PPF of 700 mmol m–2 s–1. Other thermal
environmental conditions were kept constant around the leaf area inthe window. Actinic light intensity (PPF) and short-wavelength radiation (Esx) were 350 mmol m–2 s–1 and 44.5 W m–2, 270 mmol m–2 s–1
and 34.3 W m–2, and 700 mmol m–2 s–1 and 88.9 W m–2; these parameters were measured with quantum and photosynthetically active radiation (PAR) sensors (LI-185A, LI-COR), because the wavelengths of
the actinic light were almost all in the PAR range. The spatial distributions of PPF and Esx were kept within ±5% of the mean value in the
5´5 cm window and within ±2.5% of the mean value in the 3´3 cm
area in the center of the window by adjusting the optical fiber system.
The long-wavelength radiation (Ewx) from the environment and theboundary layer conductance to heat transfer (gaHx) were determined bysolving simultaneous equations of Eq. (2) developed using dry and wet
model leaves with black felt surfaces on both sides, where the absorption coefficient (ap) of the short-wavelength radiation measured with
the LI-185A sensor was 0.84 for a kidney bean leaf and 0.98 for themodel leaf, and the emissivity (e) of long-wavelength radiation measured by the thermographic system was 0.98 for both kidney bean andmodel leaves. Consequently, Ewx and gaHx were determined to be
0.98 kW m–2 and 12.0 mm s–1 for the experiment at a PPF of 350 mmol
m–2 s–1, 0.98 kW m–2 and 14.5 mm s–1 for the experiment at a PPF of
270 mmol m–2 s–1, and 1.16 kW m–2 and 34.4 mm s–1 for the experiment at a PPF of 700 mmol m–2 s–1, respectively. The thermal environmental conditions in the measuring chamber determined the values ofEwx. The temperatures estimated from the values of Ewx at PPFs of
270, 350, and 700 mmol m–2 s–1 were 90°C, 90°C, and 105°C, respectively. The high temperatures seemed to be due to the light sources in
the measurement chamber. Boundary layer conductance to heat transfer was kept constant at each site on both sides of the leaf area by
adjusting the air current. The temperature distribution of the black felt
model leaf was maintained within 0.1°C except for the edges of thewindow.
The value of gaH can be determined by substituting Eq. (3) forEq. (1) using a wet black felt model leaf. The W in Eq. (1) was
obtained by measuring changes in weight of the wet model leaf, where
gsu and gsl in Eq. (1) were assumed to be infinity. Therefore, gaH can be
expressed with the following equation:
W ( DH ¤ DW ) 2 ¤ 3
g aH = ---------------------------------------------2 { X ( T l ) – hX ( T a ) }

(7)

1299

To assess the measurement error of the thermographic system,
we obtained the gaH by solving simultaneous equations of Eq. (2) with
input parameters determined from measurements on dry and wet black
felt model leaves (ap was 0.98. e was 0.98. gsu and gsl were assumed to
be infinity). This value was compared with gaH determined by Eq. (7)(Fig. 11). The difference between gaH obtained by solving simultaneous equations of Eq. (2) and that calculated by substituting W measured by the weighing method for Eq. (7) was within 8% regardless of
gaH values. The error in Ew obtained by the simultaneous equations of
Eq. (2) was within 1.7% of the mean value.
Simultaneous measurement of spatiotemporal variations of stomatal
conductance, NPQ, and FPSII in response to ABA treatment
Using the above-described system, we investigated the effects of
the ABA treatment on the total stomatal conductance, NPQ, and FPSII
of in situ P. vulgaris L. leaves under three intensities of actinic light.
An attached leaf mounted in an opaque cardboard frame (Fig. 10) was
horizontally placed in the measuring chamber after the controlled thermal environmental conditions were measured by using the black felt
model leaf by the methods described above. The Rst in Eq. (4) for kidney bean leaves used in this experiment was 8.2. The leaf was adapted
to the environmental conditions for 2 h, which allowed the stomatal
opening and the photosynthetic activities to reach steady state. Thiswas confirmed by low values, constancy, and uniform distribution oftemperature and chlorophyll fluorescence intensity (iF) over the leaf
area as obtained by monitoring thermal and chlorophyll fluorescence
images. Then, control images of leaf temperature and iF and iFm¢
images of chlorophyll fluorescence were measured in sequence. The
influence of the saturation light pulse used for iFm¢ measurement (as aheat pulse) disappeared within about 20 s. Thus the saturation lightpulse did not cause any changes in stomatal opening. Just after thecontrol measurement, 10–3 M ABA (Wako, 97.0% purity) was applied
to both leaf sides of a small mesophyll region (Fig. 1) with a brush.
After the treatment, a temporary decrease in leaf temperature wasobserved in the treated region, because of evaporation of the ABA
solution. This effect disappeared within 10 min with loss of the solution. Afterwards, images of leaf temperature, iF, and iFm¢ were measured at intervals of 10 or 15 min, except for the initial 15 min, for
90 min after the ABA treatment. Finally, an iFm image was measured
after the leaf area was kept in darkness for at least 1 h. Images of total
stomatal conductance, NPQ, and FPSII were calculated from the imagesof leaf temperature and the set of chlorophyll fluorescence intensity
images (iF, iFm¢, and iFm).

Acknowledgments
We thank Takateru M. Uenishi and Ryosuke Endo for helpful
discussion and comments on the manuscript. We are grateful to theJapan Society for the Promotion of Science for funding this study.

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(Received May 10, 2003; accepted September 2, 2003)