Plant Physiol. (1987) 84, 748-752
0032-0889/87/84/0748/05/$0 1.00/0

Image Analysis of Chlorophyll Fluorescence Transients for
Diagnosing the Photosynthetic System of Attached Leaves
Received for publication October 15, 1986 and in revised form March 19, 1987

KENJI OMASA*, KEN-ICHIRO SHIMAZAKI, ICHIRO AIGA, WALTER LARCHER, AND MORIO ONOE'

The National Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305, Japan (K.O., K.S., I.A.),
Institute of Botany, University of Innsbruck, A-6020 Innsbruck, Austria (W.L.), and Institute of Industrial
Science, University of Tokyo, Roppongi, Tokyo 106, Japan (M.O.)
ABSTRACTA new image instrumentation system for quantitative analysis of therapid change in intensity of chlorophyll fluorescence during dark-lighttransition (CFI, chlorophyll fluorescence induction), which is a sensitive
indicator of the various reactions of photosynthesis, was developed andits performance was evaluated. This system made it possible to resolve
CFI at any small leaf area (about 1 square millimeter) of a whole leaf
when the plant was illuminated by blue-reen light at more than 50
micromoles photons per square meter per second. In order to test theusefulness of this system, we applied it to analyze the effect of S02 on
photosynthetic apparatus in attached sunflower leaves. Dynamic CFI
imaging over the whole single leaf, where there was no visible injury,
indicated not only the local changes in photosynthetic activity but also
the site of inhibition in photosynthetic electron transport system inchloroplasts. The new instrumentation system will be useful for the
analytical diagnosis of various stress-actions on plants in situ.

transport system. Therefore, the measurement and analysis of
CFI in plant leaves in situ has been developed as a sensitive andnondestructive assay for the functional state of the photosynthetic apparatus (26, 29, 31-33). The conventional methods (27,28) give only the spatially averaged CR at a definite area because
these use point sensors. The static imaging of Chl fluorescencesuch as photography (8, 15) reveals the location of impairment
for the whole leaf in a similar manner as the DLE imaging
method, but it does not give any information involving in CFI.
We therefore developed a new instrumentation system usinga CCD image sensor for a quantitative analysis of CFI, the systemof which would give the information not only on the localized
differences in photosynthetic activity on whole leaf in situ but
also on the inhibition site in the photosynthetic system. In orderto test the usefulness of this system, we successfully applied it toanalyze the stress induced by SO2, a major phytotoxic air pollutant (10, 12, 17), on the photosynthetic apparatus of attachedsunflower leaves.

MATERIALS AND METHODS

Abiotic and biotic stresses such as air pollutants, water deficit,
high or low temperature, and virus infection cause a spatiallyheterogeneous impairment in attached leaves. The heterogeneous
impairment was indicated in stomatal responses and photosynthetic activity. For example, recent investigations employing
thermal imaging methods (9, 19, 21, 22) and DLE2 imaging
methods (6, 7) have shown evidence of spatially different responses of stomata in situ to various stresses. The DLE imaging
method, furthermore, clarified localized changes in photosynthetic activity induced by the stresses (2, 5). However, thesetechniques did not provide any information about the site ofinhibition in the photosynthetic apparatus.
Rapid changes in intensity of Chl a fluorescence during darklight transition (CFI) reflect the various reactions of photosynthesis (4, 11, 13, 18, 24), especially photosynthetic electron

Plant Materials. Sunflower (Helianthus annuus L. cv Russian
Mammoth) and cucumber (Cucumis sativus L. cv Natsusairaku
3) plants were grown in pots (10 cm in diameter and 20 cm inheight) in a controlled-environment greenhouse (1) at 25/200C
day/night temperature and 70% RH under natural lighting for 4
to 5 weeks. The pots were filled with a 4:2:4:1 (v/v) mixture ofvermiculite, perlite, peat moss, and fine gravel. The plants werewatered daily and nutrient solution was supplied every otherday. After the test plants were moved to a controlled-environment chamber designed for studies of air pollution effects onplants (1) and allowed to acclimate to the new environmental
conditions for about 5 h, attached mature leaves of the intact
plants were used in the experiments.
Image Instrumentation System for Measuring Chl Fluorescence Induction. The ordinary TV camera and recording systems
are not suited for a quantitative analysis of CFI in attached leaves
because of their low sensitivity, large afterimage, bad image
quality, AGC function, and the indistinctness in timing of theplayback image. The common tungsten and fluorescent lamps
also cannot be used as light sources for CR imaging because of
the unevenness and fluctuation in light intensity. The new image
instrumentation system (Fig. 1) was designed to overcome these
problems. A highly sensitive CCD imager with the uniformity insensitivity and the afterimage suppression was selected for a TV
camera (SONY XC-47, improved type). The image quality was
improved by the use of a computer-control VTR (SONY BVU820) with a digital time base corrector (SONY BVT-800) andthe preprocessing by using a high-speed TV image processor(KCR nexus 6400). The AGC function, which changes a rela-'Present address: Ricoh Research and Development Center, Nippa,
Yokohama, Kanagawa 223, Japan.
2Abbreviations: DLE, delayed light emission; CFI, chlorophyll fluorescence induction; TV, television; AGC, automatic gain control; CCD,
charge-coupled device; VTR, video tape recorder, ND, neutral density;
A/D, from analog signal to digital signal; I, D, P, S, M, and T, characteristic transient levels, where I is intermediary level, D is dip, P is peak,
S is quasi-stationary level, M is second maximum, and T is terminal
level; ID, DP, PS, and MT, major transient characteristics, where ID is
amplitude after subtracting the intensity image at the I level from that at
the D level (difference D - I), and DP, PS, and MT are amplitides
determined in a similar manner.
748

749

IMAGE ANALYSIS OF CHLOROPHYLL FLUORESCENCE TRANSIENTS
A| @ ~~~~~Tme Codeu

DigitalTime Bse
AGenerator/ Reader lCorrecto
Remote SwitchorComputerI

ProJectors withRemote-controShutter andFiltersI

hteImageo Optical Disc

I

Cnrle

i

T|vMonitors
and Photo
Recorder
i

TV

Vnt r

TV camera with
CCD imager
and Filters

512Hx 48d"

1.2 G1

RS-232C Computer

SystemFIG. 1.Image instrumentation system for quantitative analysis of CH
in attached leaves. A, block diagram; B, photograph. This system iscomposed of (a) a highly sensitive CCD TV camera equipped with a red
cut-off filter (Coming 2-64, >650 nmn) and an interference filter (Vacuum
optics co. Japan IF-W, 683 nm, half band width 10 nmn); (b) two
projectors of xenon lamps (Cermax LX-300F) with a remote-control
shutter, a condensing lens, a blue-green filter (Coming 4-96, 380 to 620nm), a special ND filter, and two heat-absorbing filters; (c) a computercontrol VTR with a time cede generator/reader and a digital time basecorrector, (d) a TV image processor, which is capable of high-speed
processing and 8-bit A/D conversion, with an optical disc (1.2 GB), a
photorecorder and TV monitors; and (e) a host computer system.

characteristics (ID, DP, PS, MT) were calculated by the TV
image processor on the basis of a series of images after preprocessing for shading correction and noise removal. These wereindicated by scales which correspond to the A/D conversion
level.
Measurement of Chl Fluorescence Induction in S02-Injured
Leaves. Sunflower plants were fumigated with 62.4 Mmol SO2
m 3 (1.5 ul I-') for 30 min in a growth chamber at 25.0°C air
temperature, 70% RH, and 350 grmol photons m-2 s-' lightintensity. During the fumigation, one-half of a leaf blade wascovered with a thin aluminum foil to prevent the SO2 entrance
into the plant tissue. This procedure allows the comparison
between a fumigated area (F) and an unfumigated area (UF) of
the same leaf (see Fig. 5). Immediately after the fumigation, theplant was kept in darkness in S02-free air for 30 min, then CFI
images were measured. After that CFI from the same leaf wasmeasured periodically every 60 min. The intensity of actinic
blue-green light was maintained at 125 ±6 ,umol photons m 2 S-'
on the whole leaf surface unless otherwise stated. The lightintensity was measured with a quantum sensor (LI-COR, Model
LI-185).
RESULTS AND DISCUSSION
Performance of the Image Instrumentation System. Evaluationof the system was done first. Figure 2 shows the relationshipbetween the intensity of artificial light in the likeness of fluorescence and the A/D conversion level of VTR image. The data
indicated a linear correlation between the light intensity and theA/D conversion level. Since the artificial light was sufficiently
diffused and the unevenness of the light intensity in the visualtionship between fluorescence intensity and gray level of recorded
image, was removed from the TV camera and the VTR. The
timing of the playback image was exactly defined by the use ofa shutter synchronized to the TV signal and by the time code
recorded in each image. The uneveness in intensity of light source
was improved by use of two projectors of xenon lamp (CERMAX
LX-300F) attaching a special ND filter with different density inconcentric circles.
The CRI was provoked by irradiation of the whole leaf withtwo beams of blue-green light (380-620 nm) from the projectors
with band-pass filters via shutter opening after dark adaptationof the plant for 30 min. The fluorescence image was continuously
measured at a TV field interval of Y/6o s by the TV camera
equipped with an interference filter (683 nm; half-band width,
10 nmn) and a red cut-off filter (>650 nm), and recorded withtime code by the VTR. The VTR image was digitized by a video
A/D converter after it was played hack to a still image without
guard band noise through the time base corrector. A series of thedigitized images (51 2H x 480V 8 bits) was stored on an opticaldisc (National DU- 15). A host computer system (MITSUBISHIMELCOM 70/40 and NEC PC980 1 vm4) was used to control
VTR and TV image processor.
The CRI curves, intensity images of characteristic transientlevels (I, D, P, S, M, T) and amplitude images of major transient

0.00

0.50

1.00

x10-2 jmol photons m-2s1
Light intensity
FIG. 2. Relationship between the intensity of artificial light in thelikeness of fluorescence and the A/D conversion level of VTR image.
Symbol 0 represents the mean value of A/D conversion levels of animage (512 x 480 pixels) measured at a light intensity and the verticalbar indicates ±SD. The artificial light was sufficiently diffused and theunevenness of the light intensity in the visual field of the TV camera wasmaintained within 0.1%. The light intensity was measured by a quantum
sensor through two filters placed in front of the TV camera.

750

Plant Physiol. Vol. 84, 1987

OMASA ET AL.

field of the TV camera was maintained within 0.1%, the standard
deviation of 6.3% (maximum) in the A/D conversion level was
due to image shading caused by unevenness in sensitivity of TV
camera and any noise other than guard band noise of VTR. The
image shading was corrected by calculating the ratio of an original
image to a specific image (shading master) obtained by measuring
a uniform light ofdefinite intensity because the shading occurred
mainly by lens and optical filters of TV camera. The noise wasremoved by the use of spatial smoothing filter and the averaging
of images digitized from a still VTR image (Fig. 3). For example,
the image quality could be improved to within 1% standard
deviation by the shading correction and the smoothing of 3 x 3
pixels after averaging of 10 images, when the image resolution
was 280 lines. The afterimage for the TV camera was about 4%
at 30 ms after shutter opening and decreased to 0.3% at 50 ms.
The CFI from a defined part of the cucumber leaf in situ wasmeasured by our system under different intensities of actinic
blue-green light. The CFI curves were calculated on the basis ofa series of fluorescence images (Fig. 4). Those clearly revealed
the typical IDPSMT transients (14, 24) under light intensities
from 50 to 200 ,umol photons m 2 s-' at leaf surface. In these
intensity ranges of light, we could resolve IDPSMT transientfrom any part of leaf surface which has an area of at least 1 mm2.
Fluorescence intensities of I, D, P, S, M, and T, and rates intransients of DP, PS, and MT increased as the actinic lightintensity increased. Appearance of peak P became faster with theincrease of actinic light intensity.
Because the CFI is light intensity dependent as describedabove, it is important to illuminate the whole leaf with uniform
light when we want to compare CR curves derived from different
areas (about 1 mm2) of leaf with the single whole leaf. Combination of special ND filters and two projector lamps, which wereplaced at angles of 60° and 1200 to the leaf surface, overcame
this problem. The combination kept the spatial deviation of theintensity of actinic light within 5% over a flat surface of 20 cm
diameter.
Ellenson (7) has reported a video recording system for DLE
analysis but its performance was not characterized. In general,the quality of DLE image is worse than that of Chl fluorescence
image because the image intensifier was used to image DLE. The~~~~~p4..

P>~~~~~~~~~~~
0~~~~~~~~~~~~~

100
0~~~~~~~
0C

0~~~~9

0-0

0

o

10

100
Time (s)FIG. 4. The CFI curves of a small area (about I mm2) of a healthy
cucumber leaf in situ under different intensities of actinic blue-green
light. The CFI was measured at the same definite area. Before measurement leaf was dark-adapted for 30 min. Intensity of actinic light (unit =Mmol photons m-2 s-1): (0) 200, (A) 150, (0) 100, and (A) 50. Light
intensities was varied using ND filters. Environmental conditions: air
0.1

1

temperature, 25.0°C; RH, 70%.

irradiation of a leaf with a tungsten projector lamp placed at an
oblique angle to the leaf surface (6) causes spatial differences inintensity of actinic light. Moreover, it should be noted that the
AGC function of the ordinary TV camera and VTR systemchanges a linear correlation between the intensity of DLE or CFI
and the A/D conversion level.
Effects of S02 on Photosynthetic System. Effects of SO2
fumigation on CFI in attached sunflower leaves were analyzed
by the image instrumentation system and were presented by the
CFI curves and the images with gray scale (Fig. 5). In theunfumigated leaf area (UF), the CFI clearly showed the typical
IDPSMT transients (14, 24) and almost identical transients at
any location of leaf area. Since the CFI observed upon dark-lighttransition of the leaf reflects the partial reactions of photosynthesis, we can detect the alteration in photosynthetic apparatus
by SO2 from the changes in CFI curves. As shown in Figure 5, B
140U
and C, both intensity images of characteristic transient levels (I,P, M, T) and amplitude images of major transient characteristics(ID, DP, PS, MT) in fumigated counterpart (F) strikingly differedfrom those in unfumigated area. Fluorescence intensity at I was
4.0k
300 u
raised and at P reduced markedly, and that at T was increasedin the fumigated area. Amplitude of fluorescence transients ofDP-rise and PS- and MT-decline, indicating photosynthetic acc
tivity was reduced in the fumigated leaf. The changes in theintensity and amplitude varied with the location on the leafc0
surface; the effect of SO2 was severe in locations of interveins
and veinlets than in those near large veins. Contrary to the:0 2.0laperturbation in photosynthetic apparatus shown above, there
was no visible injury in the whole surface of leaf at the end of
100 E
SO2 treatment and 2 d later.
The significance of the changes in CR induced by SO2 fumigation was as follows. Because fluorescence intensity in the early
induction phenomena is regulated by the redox state of Q, a
primary electron acceptor of PSII (4, 18, 24), the elevated I level
nrn
5
1
9
suggest that some portion of Q was brought to reduced state by
N of smoothing filter
the S02-fumigation. Since the DP rise in CFI reflects photoreFIG. 3. Improvement in image quality by image processing. The duction of Q through reductant from H20 (24, 26), a diminishedimage quality was improved by processing for shading correction and rise of DP was consistent with the inactivation of the waterspatial smoothing after averaging of images digitized from a still VTR splitting enzyme system (29). Since PS decline involves energyimage. The number of images for averaging: (0) 1, (A) 2, (0) 5, (A) 10, dependent quenching (13), the suppression of PS decline sug(U) 100, and (x) image resolution. The spatial smoothing was done by gested the depression of formation of trans-thylakoid proton
gradient probably due to the inactivation of the water-splitting
averaging of N x N pixels, where N is the size in matrix.
0

0

._

0

.

ffi

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751

IMAGE ANALYSIS OF CHLOROPHYLL FLUORESCENCE TRANSIENTS

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Time (S)

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FIG. 5. Effect Of SO2 on CRI in an attached sunflower leaf. Sunflower(15.5l1dl) at 25.0'C air
temperature, 70% RH, and 350,Mmol photons m-2 s'1 light intensity for
30 min After dark adaptation for 30 min, CRI of an attached leaf wasmeasured under 125 M~mol photons m-2 s-' actinic light. A, CRI curves
at different sites in fumigated area (F:O0, interveinal site 1; A, site 2 nearsite 3 near a veinlet) and unfumigated area (UF: 0,
a large vein;interveinal site 4). The vein and corresponding sites (I to 4) are denoted
plant was fumigated with 62.4

0.1

Mmol SO2 m3'

in a photograph of the whole leaf. B.

FIG. 6.

Recovery of CR1 from disturbances by SO2. After the meas-

urement described in

Figure 5, the same plant was kept at 25.0'C air

temperature and 70% RH in darkness and S02-free air. Then measurement of

CR1 was repeated for the same leaf every 60 min. The CR1

measured at 6 h after the initiation Of SO2 fumigation was presented. A,

CRI curves at leaf sites marked in Figure 5A. B, intensity images ofcharacteristic transient levels. C, amplitude images of major transientcharacteristics. Symbols are the same as those in Figure 5.

intensity images of characteristictransient levels (I, P, M, T). C, amplitude

images of major transientcharacteristics (ID, DP, PS, MT).

inhibition of the trans-thylakoid proton gradient formation in
addition to unidentified reactions in chioroplasts, (3, 24). Al-

enzyme system. However, the possibility that the PS decline wasthough the extent of the S02-effect on CR1 differed from area toarea on a single leaf, the mode Of SO2 action was essentially theaffected by the inhibition of electron flow from Q to PSI cannot

same.

be excluded because PS decline partly reflects the oxidation of Q

The recovery from perturbation by SO2 becomes evident bymonitoring the CR1 when the fumigated plants were allowed in

by PSI (3). Suppression of MT decline was probably due to the752
OMASA ET AL.
Plant Physiol. Vol. 84, 1987
JL, RG AMUNDSON 1982 Delayed light imaging for the earlyS02-free air (Fig. 6). In close to the large veinal area, where the 5. ELLENSON
detection of plant stress. Science 215: 1104-1106
alteration of CFI was relatively small, the CFI recovered com- 6. ELLENSON
JL, RM RABA 1983 Gas Exchange and phytoluminography ofpletely and showed the IDPSMT transient identical to that ofsingle red kidney bean leaves during periods of induced stomatal oscillations.
the unfumigated area. In most of the area near veinlets, the CR
Plant Physiol 72: 90-95

which was affected strongly, restored almost completely also. 7. ELLENSON JL 1985 Phytoluminographic detection of dynamic variations inleaf gaseous conductivity. Plant Physiol 78: 904-908
However, the fluorescence emitted from the interveinal area was 8. GIBBONSGC, RM SMILLIE 1980 Chlorophyll fluorescence photography tostill affected during this period; the peak P reappeared but its
detect mutants, chilling injury and heat stress. Carlsberg Res Commun 45:
was
still
I
and
the
level
was
elevated furthermore.
intensitylow,
269-282
The elevated I level suggests that the irreversible injury to the 9. HASHIMOTO Y, T INO, PJ KRAMER, AW NAYLOR, BR STRAIN 1984
Dynamic analysis of water stress of sunflower leaves by means of a thermal
PSII reaction center took place in chloroplasts (30). In contrast,
image processing system. Plant Physiol 76: 266-269
the elevated T level in quasi-stationary state became normal in 10. HEATH
RL 1980 Initial events in injury to plants by air pollutants. Annu Rev
this leaf area.
Plant Physiol 31: 395-431
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Photochemie der Pflanze. Biochem Z 332: 277-292
to S02-fumigation. The resistance is probably due to the smaller 12. KozIOL
MJ, FR WHATLEY, eds 1984 Gaseous Air Pollutants and Plant
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Metabolism. Butterworths, London
stomatal conductance in these areas and/or to the fast dilution 13. KRAUSE GH, J-M BRISNTAIS, C VERNOTTE 1981 Two mechanisms of reversible fluorescence quenching in chloroplasts. In G Akoyunoglou, ed, Photoof absorbed SO2 by the larger provision of water from the vein
synthesis. I. Photophysical Processes-Membrane Energization. Balaban(21, 23). Therefore, we observed that SO2 injured the leaf heterInternational Science Services, Philadelphia, pp 575-593
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J, A-L ETIENNE 1977 In vivo chlorophyll fluorescence. In J Barber,heterogeneously on the same leaf. Although the heterogeneoused, Primary Processes of Photosynthesis. Elsevier, Amsterdam, pp 203-268
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S, S ITOH, M NISHIMURA 1981 Acceleration of the decay ofwhich measures the 'averaged' CFI derived from a defined leaf 16. MORITA
membrane potential after energization of chloroplasts in intact Zea mays
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Academic Press, New Yorklution and CFR from the SO2 injury in spinach leaf macroscopi- 18. MURATA
N, M NISHIMURA, A TAKAMIYA 1966 Fluorescence of chlorophyll
cally in previous work (29). Such a partial recovery is probably
in photosynthetic systems. II. Induction of fluorescence in isolated spinach
due to the combination ('average') of the complete and incomchloroplasts. Biochim Biophys Acta 120: 23-33
19. OMASA K, F ABo, I AIGA, Y HASHIMOTO 1981 Image instrumentation ofplete recovery as indicated in the present investigation.
plants exposed to air pollutants-quantification of physiological information
As shown above, our image instrumentation system demonincluded in thermal infrared images. Trans Soc Instrum Control Eng 17:657strated not only the affected location in photosynthetic activity
663 (in Japanese and English summary) also in Res Rep Natl Inst Environ
on leaf but also the inhibition site in photosynthetic electron
Stud Jpn 66: 69-79 (1984) (in English translation).
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developed to detect injury to the photosynthetic activity (2, 5- 21. OMASAK, Y HASHIMOTO, I AIGA 1981 A quantitative analysis of the7). The method detects the injured area on the leaf in photosynrelationships between SO2 or NO2 sorption and their acute effects on plantleaves using image instrumentation. Environ Control Biol 19: 59-67
thetic activity but does not clarify the inhibition site in theK, Y HASHIMOTO, I AIGA 1981 A quantitative analysis of thephotosynthetic system because the intensity of the DLE is very 22. OMASA
relationships between 03 sorption and its acute effects on plant leaves using
similarly reduced by various factors such as inhibitions of theimage instrumentation. Environ Control Biol 19: 85-92
oxidizing and reducing sides of PSII, uncoupling of photophos- 23. OMASA K, Y HASHIMOTO, PJ KRAMER, BR STRAIN, I AIGA, J KONDO
1985 Direct observation of reversible and irreversible stomatal responses ofphorylation, anaerobic conditions, and high CO2 conditions (16,attached sunflower leaves to SO2. Plant Physiol 79: 153-158
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G. 1975 Chlorophyll Fluorescence: An intrinsic probe ofthat inhibits the oxidizing side of PSII.
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