Plant & CellPhysiol. 25(8): 1379-1388 (1984)
JSPP © 1984

Measurement of Stomatal Aperture by Digital Image Processing
Kenji Omasa 1 and Morio Onoe 2
1

Division of Engineering, The National Institute for Environmental Studies, Yatabe, Ibaraki 305, Japan
2
Institute of Industrial Science, University of Tokyo, Roppongi, Tokyo 106, JapanWe developed a new digital image processing technique for exactly measuring thedegree of stomatal opening, that is, the ratio of the width to the maximum length of a
stomatal pore, and the pore area. We applied this technique to evaluate responses
to SO2 of neighboring stomata in a small region of an intact attached leaf, with thefollowing results:
1) The pore region could be exactly extracted even when the original digital
image was of poor quality. The standard errors in the evaluation of the pore area, thepore length, and the pore width were 10 jum2, 0.3 fim and 0.3 jim, respectively.
2) A wide variety of responses to SO2 was found among neighboring stomata ina small region of an attached leaf. The differences were especially remarkable before
water-soaking and wilting. When these began, kj showed a local maximum value,
and then all the stomata began to close. These results suggest that stomatal responses
of intact growing plants to air pollutants should be examined from the standpoint of not
only the average movement of many stomata but also the movement of each stoma andthat the interrelationship among movements of neighboring stomata should be investigated to clarify the mechanism of stomatal responses to air pollutants.
Key words: Air pollutant •—• Digital image processing — Helianthus annuus —Stomatal aperture — Stomatal response.

Digital image processing has rapidly progressed with the growth of computer technology andis being widely utilized in thefieldsof television, remote sensing, and medicine (Hounsfield 1973,
Rosenfeld and Kak 1976, Castleman 1979, Onoe et al. 1981). This technique recently began
to be applied in the fields of physiological ecology and plant physiology (Hashimoto andNiwa 1978, Omasa et al. 1981a, b, c, 1983a, Hashimoto et al. 1983, Mineyuki et al. 1983, Onoe
et al. 1983, Telewski et al. 1983, Mineyuki et al. 1984, Omasa and Aiga 1985).
The stomatal aperture is generally measured with a ruler after the stomatal photographhas been taken using a scanning electron microscope and a light microscope. Since this method
involves visual observation to determine the edge of a stomatal pore, the result depends upon theexperience and knowledge of the person and inevitably includes some element of subjectivity.
Therefore, an objective instrumental technique needed to be developed by digital image
processing.
When the brightness of the stomatal pore differs remarkably from those of the guard cell andthe epidermal cell, the pore can be easily extracted from the surrounding cells by simple threshAbbreviations: la, width of a stomatal pore; lb, length of a stomatal pore; lbmam 'b of an open stoma, i.e.,
maximum value of lb; kj, degree of stomatal opening expressed by lo/lbmai; A r , area of a stomatal pore; SIT,
silicon intensifier target; FSS, flying spot scanner; RH, relative humidity; ppm (v/v), parts per million in volume
units.
1379

1380

K. Omasa and M. Onoe

olding of the original image, which is a digital image processing technique. Hashimoto et al.
(1983) recently evaluated the pore area of stomata observed with a scanning electron microscopeby this method. However, since many stomatal images, especially those observed with a lightmicroscope, do not satisfy the above condition and the edge of the stomatal pore is not clear, thesimple thresholding cannot be used for pore extraction. Also, there has been no report on theobjective evaluation of pore width and pore length, which are used as the common indicator of
stomatal aperture.
We, therefore, developed a new digital image processing technique for exactly measuring
the degree of stomatal opening, that is, the ratio of the width to the maximum length of a stomatalpore, and the pore area as indicators of stomatal aperture. We also applied this technique toevaluate the responses to SO2 of neighboring stomata in a small region of an intact attached
leaf.
Materials and Methods
Plant materials—Sunflower (Helianthus annuus L cv. Russian Mammoth) plants were grown inan environment-control greenhouse (Aiga et al. 1982) at 25/20°C day/night temperature and 70%
RH under natural light for 4 to 6 weeks after being sown in pots. The pots were filled with a
4 : 2 : 4 : 1 (v/v) mixture of vermiculite, perlite, peat moss, and fine gravel, which was moistenedwith nutrient solution. The plants were irrigated daily. After the test plants had been moved
to an environment-control chamber designed for studies of air pollution effects on plants (Aigaet al. 1982) and acclimatized to the new conditions, mature leaves of the intact plants were usedin the experiments.
Observation of stomatal responses to air pollutants—Stomatal responses of intact growing plants to

SO2 and O3 were continuously observed using a remote-control light microscope system (Omasa
et al. 1983b). A light microscope with a wide working distance (about 13 mm) at high magnification (a 50 x objective, 1.5 x and 2x amplifiers and a TV adapter lens) and an SIT camera
head with high sensitivity were set in the chamber. An attached leaf was fastened to a movablemicroscope stage and illuminated until its stomatal apertures attained a steady state, after which
the leaf was treated with air pollutants. During the experiments, the microscope image wasprojected onto a TV monitor outside the chamber, giving about 800 X to 1,600 X magnification
with high resolution and little distortion. The images were recorded photographically on blackand-white negative roll film for later study. Camera sensitivity, microscope focus, and movement of the microscope stage were adjusted by remote controllers outside the chamber. Air
temperature and relative humidity in the chamber and around the leaf were maintained at 25.0°C
and 60%. The leaf was illuminated with tungsten lamps at about 600 jitE-m"2^-1 with the light
passing through heat-absorbing filters.
Digital image processing system—The stomatal image photographed on the negative film wasconverted with an FSS into a digital image (256H X 256V, 256 gray levels), which was analyzed by
a computer system with a monochromatic TV monitor for image display.
Principle and procedure of image processing—Fig. 1 shows the flow diagram of digital image
processing for evaluating the pore width, the pore length and the pore area as indicators of
stomatal aperture.
The original digital image converted by the FSS involved scanning lines of the TV monitor.
The brightness of the stomatal pore in the image did not differ sufficiently to allow thresholding
from those of the guard cell and the epidermal cell, and the edge of the pore was not clear.
Therefore, filtering by discrete Fourier transform and unsharp masking were used to remove theTV scanning lines and to enhance the image as a first step of the image processing.

Measurement of stomatal aperture

1381

Original digital image(256Hx 256V 256 gray levels)

I (a)Discrete Fourier transform

Multiplying a Hanning filter (c)to remove TV scanning lines

Multiplying a Hanning filter (g)to make a blurred image||

Discrete inverse
Fourier transformDiscrete inverse
Fourier transform

(e)

(i)

*i

[Thresh olding]

Unsharp masking

11(0Thresholding[Filling up small holes]

I

t

1

Evaluation of pore area

Extraction of stomatal pore

I

(1)

I

Edge detection

Computation of principal components

1Overlapping pore edge with original image

1 /m\

Shift and rotation of coordinates
Evaluation of length and width of poreFig. 1 Flow diagram of digital image processing for evaluating width, length and area of a stomatal pore.

Withf(x, y) as the gray level at the pixel (x, y=0, 1,2, •••,N—1) in the original digital image,the two-dimensional discrete Fourier transform F(u—N/2, v—N/2) off(x, y), where the origin
after the transform was moved to the center of frequency square, was given by
F(u-N/2,v-N/2)=1SrE1
IN

x =o

S 1 (-l) x+y f(x,y)exp[-j27r(ux+vy)/N]
y=0

for u, v=0, 1,2, —,N —1.
After multiplying F(u—N/2, v—N/2) by the Hanning filter H(u—N/2, v—N/2, fc) toremove the TV scanning lines or to obtain a blurred image for the unsharp masking, each
inverse Fourier transform g(x', y', fc) could be expressed byg ( x ', y ',f c )=* (_1)*+"NN£
IN

u =o

u+v
s'(-l)' ( ) u + vH(u-N/2,v-N/2,f c )v =0xF(u-N/2,v-N/2)exp[j27r(ux+vy)/N]
for x'=x—N/2 and y'=y—N/2, where

K. Omasa and M. Onoe1382

if fuvS:fc
H(u-N/2,v-N/2,f.) =if fu,<fc
andfuv = [ ( u - N / 2 ) 2 + (v-N/2) 2 ] 1/2
The parameter fc of the Hanningfilterwas determined according to the above purpose. The
unsharp masking, in which the blurred image g(x', y', fc2) ( (i) in Fig. 1) was subtracted fromimage g(x', y', fci) (e) after removal of the TV scanning lines, had the effect of increasing the
average ramp steepness and of increasing the contrast just at the pore edge. The unsharp
masked image gu(x',y') was given byg.(x', y')=g(x', y', f«i)-g(x', y', fc2)
The pore region of appointed stoma could be extracted from the neighboring cells by thresholding gu(x', y'). The result could be expressed as the two-valued image gT(x', y').
if gu(x', y') > T at appointed stoma
otherwise
where T is a threshold. The threshold was determined to be about zero in the gray level of theunsharp masked image and to give the maximum area in the pore region extracted elliptically.
When there were small holes in the threshold region due to poor quality of the original image(although this was unusual), the holes were completelyfilledup.
The area Ar of the stomatal pore was obtained by counting pixels in the extracted region.
A r =k 2 A
where
N/2—1

A=

2

N/2—1

E gT(x'.y')x'=-N/2 y'=-N/2

and k is a coefficient for correcting the magnification.
The pore width U and the pore length U were evaluated from frequency distributions to two

Fig. 2 Conception for evaluating width and length of a
stomatal pore.

Measurement of stomatal aperture1383

rectangular principal components of the extracted region (Fig. 2). The principal components
X, Y were defined by
X = (x' -x 0 )cos 8+ (y' -y o )sin 8
andY = - (x'-x o )sin 9+ (y'-y 0 )cos 8
where
1

N/2-1

N/2-1

xo=-r- S

2 x'gT(x',y')Ax' = _N/2 y'=-N/2

1

N/2-1

yo=TA

N/2-1

2

2 y'gT(x',y')«'=_N/2

N/2-1

y'=-N/2

N/2-1

x= 2

2

(x'-xo)*gT(x',y')x'=-N/2 y'=-N/2
N/2—1 N/2—1

and
N/2-1

N/2-1

xy= 2

2

x"=-N/2(x'-xo)(y'-yo)gT(x',y')y'=-N/2

With P(X) and P(Y) as the frequency distributions to principal components X, Y, they were
evaluated by computer after the shift and rotation of coordinates from x' and y' to X and Y, andthen la and lb were determined fromla = k ( Y 4 - Y 3 )lb=k(X4-X3)
where Xi to X4 and Yi to Y4 were defined by
P(Yi)=P(Y a )=O
2 P ( X ) = 2 P(X)=0.005A
x=x,
x=x.

X4>X3

2 P ( Y ) = S P(Y)=0.005A

Y 4 >Y 3

and
y=Y,

Y=Y.

The degree of the stomatal opening k, was expressed by the ratio la/lbmax, where Umax was
the length lb of the stomatal pore of an opened stoma, that is, the maximum value of lb. Theedge of a stomatal pore could be detected from gT(x', y') by a simple difference operator.

Results and Discussion
Fig. 3 shows the sequence of the digital image processing for evaluating the stomatal aperture according to the flow diagram in Fig. 1. The original digital image (a) involved TV

138*

K. Omasa and M. Onoe

Measurement of stomatal aperture

1385

Table 1 Accuracy in the evaluation of area, length, and width of a stomatal pore
Area

LengthWidthMean value

433

34.1

16.4 /imStandard error

10

0.3

0.3

scanning lines of vertical black-and-white stripes. Being clear and regular, the lines appeared as
specific frequency components with sharp peaks at u~ ±68 in the Fourier spectrum (b)|F(u— N/2, v—N/2)| obtained from the magnitude of each of the transform terms. These
components differed remarkably from low-frequency components with the information for thestomatal form. Therefore, the TV scanning lines were easily removed by a Hanning filter of
fc=60 (c), which was a low pass filter with the restraint function of frequency extension and leakage. The Fourier spectrum after the filtering and its inverse transform image are shown in (d)and (e), respectively. Since the brightness of the stomatal pore did not differ from that of the guard
cell or the epidermal cell, the pore region could not be extracted by the simple thresholding (f).
Thus, the unsharp masking was used for image sharpening. The blurred image (i) for theunsharp masking was obtained by the inverse transform of a Fourier spectrum (h) after multiplication with a Hanning filter of fc=10 (g). The unsharp masked image (j) increased in contrast
at the pore edge and in differences of brightness between the pore and its neighboring cells.
Therefore, the pore region of the appointed stoma could be easily extracted from the neighboring
cells after the thresholding; (k) was a two-valued image after the thresholding and (1) was a poreregion extracted from the image (k). From image (m) obtained by overlapping the pore edge
with the original image, we confirmed that the pore region had been exactly extracted. The
pore area was computed from the extracted image (1). The pore width and the pore length were
evaluated after the shift and rotation of coordinates to the principal components of the poreregion; (n) expressed principal components and (o) the shift and rotation of the coordinates.
The quality of the original digital image depends upon the display image itself, the development of the film, the FSS conditions and other factors, and affects the accuracy of the stomatal
aperture evaluation. Therefore, the accuracy was first examined using ten original images withthe same stomatal aperture but with differences in quality and also using continuous images ofro 0.8

400«

>enii

r-

t

I5

° 0.6

300 <
4)

ID

c

Jo.4

kl

<>
/
Fig. 4 Changes in A r and k) of a stoma during

O

- Ar
\
.0__o-o-o-°x \

o
v 0.2

t
°3
1 ppm

£,

a
200 ra

\o\ e
\V I

Q
n 0

-10

0

f

100 2
Q»-

0

10
20
30
40
Exposure time (min)

U
^*
n tf

,_Q
y—^^—

^ Q _ .

O3 exposure.

I0

\\l
Q

IS

s

Fig. 3 Sequence of digital image processing for evaluating stomatal aperture according to theflowdiagram of Fig. 1.
(a) to (o) correspond to those in Fig. 1. The coordinates are shown in (a) for (x, y) and (b) for (u, v).

K. Omasa and M. Onoe

13$6

d

e

f

Fig. 5 Original digital images overlapped with the pore edge at the time points (a to f) in Fig. 4.

different quality during 1 ppm (v/v) O3 exposure. Table 1 shows the standard errors in theevaluation of pore area, pore length, and pore width of the former ten stomatal images. The
errors of the area, the length, and the width were 10 /jm2, 0.3 |im and 0.3 /im, respectively. Fig.
4 shows the changes in the pore area Ar and the degree kj of opening of a stoma during the O3
exposure. The slow increase of both Ar and k, began from before the exposure and continued
until 10 min (b) after its start. Thereafter, both began to decrease rapidly and reached a constant level at about 25 min (e). Fig. 5 shows original digital images overlapped with the poreedge at the time points (a) to (f) in Fig. 4. They confirmed that the pore region had been exactly extracted even when the quality of the original image was poor, thus displaying the accuracyof the results.
In the second half of this study, we examined the responses to SO2 of neighboring stomata
in a small leaf region of an intact growing plant. Fig. 6 shows a microphotograph of a small
region (about 320 X 320 yttm2) in which six stomata were observed. Fig. 7 shows the changes inthe degree k, of the opening of these stomata during the 2 ppm (v/v) SO2 exposure. These
stomata showed almost uniform and constant k, until about 20 min after the start of the exposure, and then a wide variety of stomatal movements began; the largest value in k, was about
twice as large as the smallest value at 45 min and became about three times as large at 90 min.
Water-soaking and wilting began to appear in the subsidiary cells at about 55 min, when k, wasa local maximum value, and then all the stomata began to close. This phenomenon was assumed
to be caused by increased water loss from the subsidiary cell due to SO2, which affects the membrane and osmotic pressure, with a difference resulting in the turgor between the guard cell and thesubsidiary cell (Heath 1980, Unsworth and Black 1981). However, the explanation of guard
cell behavior is not the primary objective of this paper. This phenomenon will be examined morethoroughly in a future study.
Changes in stomatal conductance or stomatal resistance of a small leaf region to air pollutants
have been investigated using a porometer and a thermal instrumentation system (Unsworth andBlack 1981, Omasa et al. 1981b, c). The latter system could show changes in spatial stomatal

1387

Measurement of stomatal aperture

30
60
90
Exposure time (min)50/urnFig. 7

Fig. 6

Fig. 6 Microphotograph of a small region of an intact attached leaf.
Fig. 7 Changes in ki of stomata (a tof) in Fig. 6 during SO2 exposure. Small arrows (t or J) show when watersoaking and wilting began to appear.

resistances over an attached leaf and differences have been noticed in the change of the resistanceat each leaf site (Omasa et al. 1981b, c). Also, transpiration, which is an indicator of stomatal
movements, of a whole plant and one leaf to air pollutants is known to vary with the species, age,
growing environment, air pollutant concentration, and other factors (Kondo and Sugahara 1978,
Omasa and Abo 1978, Omasa et al. 1979, Furukawa et al. 1979, Unsworth and Black 1981).
However, since stomatal resistance and transpiration indicate the average movement of many
stomata, they cannot give the responses of each stoma to air pollutants.
In the present study, we showed for the first time the wide variety of responses to SO 2 ofneighboring stomata of an attached leaf. Our findings suggest that the stomatal responses ofintact growing plants to air pollutants should be examined not only by studying the average
movement of many stomata but also by examining the movement of each stoma and by investigating the interrelationship among movements of neighboring stomata in order to clarify themechanism of stomatal responses to air pollutants. Our remote control microscope system(Omasa et al. 1983b), developed to study individual stoma, can be used with the new image
processing technique described in this paper to precisely evaluate the degree of stomatal opening
and the pore area. Even with images of poor quality, this technique is very effective.
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(Received June 4, 1984; Accepted August 23, 1984)