Annals of Botany 87: 631±639, 2001
doi:10.1006/anbo.2001.1386, available online at http://www.idealibrary.com on
Epicuticular Phenolics Over Guard Cells: Exploitation for in situ Stomatal Counting by
Fluorescence Microscopy and Combined Image Analysis
G E O R G E K A R A B O U R N I OT I S * , D E S P I N A T Z O B A N O G LO U , DI M O S T H E N I S
N I KO LO PO U LO S and G E O R G I O S L I A KO PO U LO S
Laboratory of Plant Physiology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera
Odos 75, 11855 Botanikos, Athens, Greece
Received: 4 October 2000 Returned for revision: 28 December 2000
Accepted: 22 January 2001 Published electronically: 26 March 2001
Guard cells emit an alkali-induced, blue ¯uorescence upon excitation by ultraviolet radiation (emission maximum
energy at 365 nm). Fluorescence emission of guard cells was brighter than that of the neighbouring epidermal cells in
a number of wild and cultivated plants including conifers, but the relative ¯uorescence intensity and quality was
species-dependent. Three representative plants possessing stomatal complexes which di€ered morphologically were
studied: Olea europaea, Vicia faba and Triticum aestivum. Immersing leaves of these plants in chloroform for 30 s
(thereby removing epicuticular waxes) signi®cantly reduced the intensity of the ¯uorescence emitted by guard cells.
This indicates that guard cell ¯uorescence could be due to either an increased concentration of ¯uorescing compounds
( probably wax-bound phenolics), or a thicker cuticular layer covering the guard cells. Given that the alkali-induced
blue ¯uorescence of the guard cells is a common characteristic of all plants examined, it could be used as a rapid and
convenient method for in situ measurements of the number, distribution and size of stomatal complexes. The
proposed experimental procedure includes a single coating of the leaf surface by, or immersion of the whole leaf in, a
10 % solution of KOH for 2 min, washing with distilled water, and direct observation of the leaf surface under the
¯uorescence microscope. Fluorescence images were suitable for digital image analysis and methodology was
# 2001 Annals of Botany Company
developed for stomatal counting using Olea europaea as a model species.
Key words: Cuticle, epicuticular waxes, ¯uorescence microscopy, image analysis, phenolics, stomata.
I N T RO D U C T I O N
Stomata regulate the exchange of water vapour and CO2
between the plant and the atmosphere, mainly through
changes in aperture of the stomatal pore. Therefore,
stomata play a pivotal role in controlling the balance
between water loss and carbon gain. For this reason, guard
cells are of great importance in plant biology. Moreover,
direct or indirect methods of measuring the number and
dimensions of stomata have played an important role in
many studies of anatomical, physiological, ecological and
agricultural interest.
Fluorescence microscopy has been successfully applied to
investigate the distribution of phenolic compounds in plant
cells and tissues (Rost, 1995). Under the appropriate
conditions of excitation, several classes of phenolic compounds can be visualized either by auto¯uorescence or by
the use of speci®c compounds that induce the ¯uorescence of
these substances (Harborne, 1989; Rost, 1995; Karabourniotis and Fasseas, 1996; Karabourniotis et al., 1998).
Fluorescence microscopy has been used for the detection of
¯avonoids and other related phenolics in the vacuoles of
guard cells of Allium, Vicia and Pisum (Zeiger and Hepler,
1979; Palevitz et al., 1981; Schnabl et al., 1986; Weissenboeck et al., 1987), and in the peristomatal protuberances of
the grape berry (Blanke et al., 1999), using blue light to emit
¯uorescence.
* For correspondence. Fax 003 1 5294286, e-mail [email protected]
0305-7364/01/050631+09 $35.00/00
The aim of the present study was to investigate the cause
of the di€erences in ¯uorescence emission between guard
cells and neighbouring epidermal cells under UV excitation.
In addition, the occurrence of this phenomenon in a number
of wild and cultivated plants was investigated. We conclude
that ¯uorescence microscopy o€ers a new, simple, convenient and time saving method for measuring the number
and dimensions of stomata. Moreover, the eciency of an
image analysis method to extract data from such images was
evaluated. Digital processing of biological systems o€ers
quick, accurate and unbiased data acquisition and analysis.
M AT E R I A L S A N D M E T H O D S
Plant material
Leaves from a number of plant species (Table 1) were
collected during spring and summer 1998 from the
experimental plantation of the Agricultural University of
Athens, Greece. Plant material was wrapped in plastic bags
and transferred immediately to the laboratory. Leaves of
Olea europaea were dehaired using self-adhesive tape prior
to observations to remove the trichome layer that emits
auto¯uorescence (Karabourniotis and Fasseas, 1996; Karabourniotis et al., 1998) and prevents contact of the leaf
surface with the alkaline solutions. The trichome layer also
completely covers the abaxial surface of the leaves and
prevents visualization of stomata. Arabidopsis thaliana,
Commelina communis and Tradescantia sp. were grown in
# 2001 Annals of Botany Company
632
Karabourniotis et al.ÐFluorescence Microscopy of Stomata
a growth chamber under a 16/8 h (day/night) photoperiod
and a temperature of 24/188C (day/night). Light intensity
was 250 mmol m ÿ2 s ÿ1.
Fluorescence microscopy
Observations were made using a Zeiss Axiolab ¯uorescent microscope equipped with a G-365 excitation ®lter
and an FT-395 chromatic beam splitter.
The leaf surface was observed directly under the
microscope using either untreated leaves, or those previously immersed in a 10 % (w/v) solution of KOH for
2 min and then subsequently washed with distilled water.
This treatment permits stomatal counting by simple
observation under the microscope.
Image analysis
A video camera (CCD colour camera SSC-DC 38P/45,
SONY Corporation, Tokyo, Japan) was attached to the
microscope. Images of the abaxial surface of dehaired leaves
of Olea europaea, untreated with alkaline solution, were
captured in a PC using a video board (Pinnacle PCTV,
Pinnacle Systems GmbH, Braunschweig, Germany) as 24bit RGB with a resolution of 640 480 pixels, converted to
8-bit grey scale and stored in Tagged Image File Format
(TIFF).
Due to non-uniform illumination of the images (which is
quite frequent in ¯uorescence microscope images obtained
from fresh leaves), we developed an Illumination Correction
Filter (ICF) using MatLab Software (version 5.1.0.421,
Mathworks Inc.). The program listing is available from the
authors on request. It provides a useful tool which permits
rapid preparation of the image for data extraction. The ICF
makes the illumination of the image uniform by automatically computing the mean intensity of the background in
several areas of the image by using a prede®ned grid, and
uses these values to standardize the image background
(Russ, 1999). Brie¯y, ICF produces 100 sub-images by
dividing the input image into 10 by 10 parts, and computes
the mean of the minimum grey scale values for each one of
the 100 sub-images. Each mean is used to yield a correction
factor based on the di€erence between each sub-image's
mean and the mean value of the whole image. Then, using
an algorithm, ICF shifts the illumination of each sub-image
based on the corresponding correction factor. As a result,
the intensity of a sub-image that is darker (or brighter)
compared to the mean intensity of the whole image, will
shift to higher (or lower) values (Fig. 5A). Using another
algorithm, the shifted intensity frequency plot of each subimage is stretched to match the intensity frequency plot of
the whole image (Fig. 5A) and the image is composed again.
Images corrected with ICF were processed with ImagePro Plus, version 3.01 (Media Cybernetics) executing a
macro command which carries out the following actions:
(a) applies the median ®lter (7 by 7 pixels; strength 10;
three passes); (b) applies the background ®lter (dark
background; feature width 20 pixels); (c) subtracts the background output from the image; (d) converts the backgroundcorrected image to binary using a threshold interval of 23 to
255 (white on black); and (e) counts the image objects. The
median ®lter removes the random noise from epidermal
cells and stomatal complexes, without changing either the
illumination di€erences across boundaries or the shape and
size of the image objects (Russ, 1999). The background
subtraction enhances the image by improving object
discrimination (Anonymous, 1998). Counting uses a single
class variable classi®cation based on object area. The
classi®cation of the counted objects was made by using two
bins (bin #1: ten±180 pixel2 and bin #2: 180±1000 pixel2).
First class objects are classi®ed as stomatal complexes and
second class objects as trichome bases.
Method evaluation and adaptation to di€erent species
Images from ten individual dehaired, intact olive leaves
were processed to assess the accuracy and reproducibility of
the method. The surface covered by each image was
0.308 mm2. Stomata and hair bases were ®rst counted
manually on the screen, and then the same images were
processed using the image analysis method described.
Finally, the number of stomata and hair bases was expressed
per surface unit (objects mm ÿ2).
The method described above was also applied to a
di€erent sample (abaxial surface of Vicia faba leaves) by
minor modi®cations of the processing steps: (a) the
extraction of the blue channel from the initial RGB image
instead of converting the image to grey scale; and (b)
selecting a di€erent threshold interval (45 to 255). All
objects were counted as stomatal complexes.
Estimation of the relative ¯uorescence intensity emitted by
stomatal complexes
To measure the relative intensity of the emitted ¯uorescence, the colour information of the images was discarded
using standard image processing. The camera response
between ten±170 (grey scale values in the range of the linear
response of the camera) was used (Karabourniotis, 1998).
Comparison of measured stomatal apertures between
methods
Seeds of Vicia faba were sown directly into pots
outdoors. Four weeks after sowing the plants were divided
into two groups. One group of plants was well watered,
while the second group received no water supply for several
days. Twenty mature leaves from each group were chosen.
A stomatal impression was taken from one half of each leaf,
while the other half of each leaf was observed immediately
under the microscope according to the protocol given
above. Leaf impressions were taken by standard methods
(Weyers and Meidner, 1990; Bolhar-Nordenkampf and
Draxler, 1993).
Other measurements
Epicuticular material was removed by immersing intact
leaves in chloroform (Wollenweber, 1985) for 30 s.
Karabourniotis et al.ÐFluorescence Microscopy of Stomata
R E S U LT S A N D D I S C U S S I O N
The observation that guard cells of Olea europaea emitted a
blue auto¯uorescence under UV light (Fig. 1A) was the
origin of the present experimental work. The initial working
hypothesis was formulated on the basis that this ¯uorescence was due to lignin deposition on the cell walls of the
guard cells; it is known that the guard cells of gymnosperms
and some ferns contain lignin (Willmer, 1983; Fahn, 1990).
Lignin gives a strong blue ¯uorescence under UV excitation,
which turns greenish upon the addition of alkali (Rost,
1995). Blue ¯uorescence in cell walls may also be attributed
633
to the occurrence of ferulic acid (Harris and Hartley, 1976;
Lichtenthaler and Schweiger, 1998), which is also responsible for the blue-green ¯uorescence emitted by the
epidermis of sugar beet leaves (Morales et al., 1996).
Treatment with alkaline solution caused guard cells of
olive to ¯uoresce more brightly than untreated controls
(Fig. 1B). However, histochemical staining with phloroglucinol-HCl, which detects lignin (Johansen 1940), gave a
negative result (data not shown), indicating that ¯uorescence was not due to lignin. Alkali treatment also induced
the emission of blue ¯uorescence from epidermal cells
F I G . 1. Fluorescence micrographs of the abaxial surface of O. europaea (A±C) and V. faba (D±F) leaves after di€erent treatments. A and D,
Untreated leaves. Arrows in A show trichome bases (trichomes have been removed, see Materials and Methods). B and E, leaves immersed for
2 min in a solution of 10 % KOH and washed with distilled water. C and F, Leaves immersed for 2 min in a solution of 10 % KOH, washed with
distilled water and immersed in chloroform for 30 s. In C and F note the removal or reduction of the ¯uorescence emitted from the guard cells.
Bars ˆ 50 mm.
634
Karabourniotis et al.ÐFluorescence Microscopy of Stomata
F I G . 2. Fluorescence micrographs of the abaxial leaf surfaces of T. aestivum (A±C) and P. halepensis (F and G), as well as from cross-sections of
O. europaea (D and E) leaves. A, Untreated leaf. B, Leaf immersed for 2 min in a solution of 10 % KOH and washed with distilled water. C, Leaf
immersed for 2 min in a solution of 10 % KOH, washed with distilled water and immersed in chloroform for 30 s. In C note the decrease in
¯uorescence emitted from the guard cells. Arrows in D and E show cuticular ledges over guard cells in O. europaea leaves. Arrowheads show the
thin cuticular layer covering the cell walls of the guard cells in the substomatal cavity. D, Dehaired leaf immersed for 2 min in 10 % KOH. E,
Dehaired leaf immersed for 2 min in a solution of 10 % KOH, washed with distilled water and immersed in chloroform for 30 s. Sections cut after
this treatment. Note the decreased intensity of the ¯uorescence emitted from cuticule and the absence of ¯uorescence from the cuticular ledges. F,
Leaf of P. halepensis immersed for 2 min in a solution of 10 % KOH and washed with distilled water. G, Leaf immersed sequentially in
chloroform for 30 s, in a solution of 10 % KOH for 2 min and washed with distilled water. Bar ˆ 50 mm (A±C, F and G) and 8 mm (D and E).
(Fig. 1B). This showed that the ¯uorescing compound(s) is
also localized in the epidermis, probably in lower concentrations or of a di€erent type to that in the guard cells.
Cross-sections of olive leaves showed that the cuticle was
the main structure to emit blue ¯uorescence (Fig. 2D). This
blue ¯uorescence was also emitted from the cuticular ledges
covering guard cells (Fig. 2D). Phenolic compounds (e.g.
hydroxycinnamic acids such as p-coumaric acid and ferulic
acid, as well as ¯avonoid aglycones) are common constituents of the cuticle and, in a number of species, are extruded
onto the surfaces of leaves (Riley and Kolattukudy, 1975;
Kolattukudy, 1980; Wollenweber, 1985; Barnes and
Cardoso-Vilhena, 1996). Therefore, it was possible that
the blue ¯uorescence emitted by the guard cells of
Karabourniotis et al.ÐFluorescence Microscopy of Stomata
200
180
Grey scale value
160
140
120
100
80
60
40
0
5
20
10
15
KOH concentration (%)
25
F I G . 3. The relative intensity (as grey scale values) of the induced
¯uorescence emitted from the guard cells of O. europaea leaves as a
function of the concentration of KOH (see Materials and Methods).
Leaves were immersed in the particular solution of KOH for 2 min.
Five leaves were used and the maximum grey value from ten guard cells
on each leaf was recorded. Values are means, bars indicate s.d.
220
200
180
Grey scale value
O. europaea was due to the occurrence of certain phenolic
compounds in the cuticular layer covering them. To con®rm
that the ¯uorescing compounds were deposited in the
cuticular layer, olive leaves were immersed in chloroform
for 30 s to remove epicuticular waxes. This treatment
caused a signi®cant reduction in the intensity of the blue
¯uorescence emitted from the guard cells (Fig. 1C). In
cross-sections, the ¯uorescence derived from the cuticular
ledges almost disappeared (Fig. 2E).
Similar results were obtained using leaves of two other
representative plant species with morphologically di€erent
stomatal complexes, Vicia faba and Triticum aestivum.
Alkali treatment induced blue ¯uorescence mainly from
guard cells, whereas this ¯uorescence disappeared upon
immersion in chloroform (Figs 1D±F and 2A±C). It seems
probable, therefore, that the observed di€erences in patterns
of ¯uorescence emission between guard cells and neighbouring epidermal cells, might be caused by increased deposition
and/or di€erent composition of ¯uorescing compounds in
the cuticular layer covering guard cells. Alternatively, the
greater thickness of the cuticular layer over these cells might
be responsible for this phenomenon. In T. aestivum
(Fig. 2B), as well as in other monocots examined in the
present study (Commelina communis, Tradescantia sp. and
Zea mays), the ¯uorescence emitted seemed to be derived
from epicuticular material over the anticlinal cell walls of the
guard and epidermal cells, and not from the whole surface of
the guard cells, as in other species (Fig. 2B).
The ¯uorescence emission characteristics of the chloroform washes of the leaves of Olea europaea and Prunus
persica concur with the ¯uorescence emission pattern of the
corresponding leaf surfaces. High performance liquid
chromatographic (HPLC) analyses of the rinses showed
the occurrence of wax-bound phenolic compounds, ferulic
acid being the main ¯uorescing component (Liakopoulos
et al., 2001).
The relative intensity of the induced ¯uorescence emitted
by the guard cells of O. europaea leaves depended on the
concentration of KOH and the incubation period (Figs 3
and 4). A concentration of 10 % KOH applied for 2 min
was sucient for the satisfactory induction of the blue
¯uorescence.
The blue auto¯uorescence of guard cells was not observed
in all plant species examined (compare Fig. 1A with Figs 1D
and 2A, see also Table 1). However, the blue, or blue-green
alkali-induced ¯uorescence of guard cells, brighter than that
of the neighbouring epidermal cells, was a common
characteristic of all plants examined, including species of
special value for stomatal research, such as Commelina
communis, Tradescantia sp., Vicia faba and Arabidopsis
thaliana. The relative ¯uorescence intensity emitted from the
guard cells was species-dependent (Table 1). In coniferous
species such as Pinus halepensis and P. pinea, ¯uorescence
emission induced by the alkaline solution was apparent only
after the immersion of leaves in chloroform (Fig. 2F and G).
This could be explained by the fact that the stomata of a
number of coniferous species are sunken and waxes ®ll the
stomatal antechamber (Juniper and Je€rey, 1983; Willmer,
1983). Chloroform treatment removed the epicuticular
waxes above the stomatal apparatus and, as a result, blue
635
160
140
120
100
80
60
40
0
1
2
3
4
5
6
Time (min)
F I G . 4. The relative intensity (as grey scale values) of the induced
¯uorescence emitted from the guard cells of O. europaea leaves as a
function of the time of immersion in a solution of 10 % KOH (see
Materials and Methods). Five leaves were used and the maximum grey
value from ten guard cells of each leaf was recorded. Values are means,
bars indicate s.d.
¯uorescence emission from the guard cells of both
coniferous species was apparent upon treatment with alkali
(Fig. 2G). Lignin deposition in guard cell walls of conifer
leaves (Willmer, 1983; Fahn, 1990) could be responsible for
the observed blue ¯uorescence, since the chloroform
treatment probably removed all epicuticular materials
from the leaf surface and the KOH solution was able to
reach the lignin of the cell walls.
Upon excitation with blue light, guard cells of onion
(Allium sp.) emit a green auto¯uorescence with emission
636
Karabourniotis et al.ÐFluorescence Microscopy of Stomata
T A B L E 1. Relative intensity of the emitted ¯uorescence from
guard cells of untreated (ÿKOH) and alkali treated
(‡KOH) leaves (see Materials and Methods) of a number
of plant species, viewed at the same magni®cation (20)
(‡) KOH
‡
ÿ
‡
‡‡
‡
‡
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
‡‡
‡
‡
ÿ
‡
‡
ÿ
ÿ
‡
‡
ÿ
‡
‡
‡‡
‡‡‡
‡‡
‡‡‡
‡‡
‡‡
‡‡
‡‡
‡
‡‡‡
‡‡‡
‡
‡‡‡‡
‡‡
‡
‡‡‡
‡‡
‡‡‡
‡‡‡
‡
‡‡‡
‡‡‡
‡
‡‡‡
‡‡
Frequency
Anethum gravendens
Arabidopsis thaliana
Beta vulgaris
Citrus limonia
Citrus mobilis
Commelina communis
Cydonia oblonga
Eriobotrya japonica
Ficus carica
Juglans regia
Lens culinaris
Morus alba
Olea europaea
Origanum vulgaris
Pistacia vera
Prunus amygdalus
Prunus avium
Prunus communis
Pyrus domestica
Salvia fruticosa
Tradescantia sp.
Triticum aestivum
Vitis vinifera
Zea mays
Zizyphus sativa
(ÿ) KOH
B
Frequency
Species
A
The number of crosses represents the relative intensity of the emitted
¯uorescence (See Materials and Methods): grey scale values range:
‡‡‡‡, 4150; ‡‡‡, 150±120; ‡‡, 120±80; ‡, 80±50; ÿ, 550.
maximum at 520 nm, (Zeiger and Hepler, 1979; Palevitz
et al., 1981). A similar ¯uorescence was also observed in
Vicia and Pisum after treatment with alkali (Schnabl et al.,
1986). Weissenboeck et al. (1987) suggested that the
¯uorescence might be due to the presence of ¯avonoids in
the vacuoles of guard cells. Our results for three species
suggested that the observed auto- or induced ¯uorescence
from the guard cells under UV light (Figs 1C, F and 2C),
was derived mainly from epicuticular material. Under the
current excitation and observation conditions, there was no
indication that the ¯uorescence emission was derived from
substances located in the vacuoles of guard cells. When
leaves were immersed in chloroform followed by KOH,
guard cells of a number of plants emitted a yellow-green
¯uorescence (data not shown), possibly due to the presence
of ¯avonoids and other related compounds in the protoplast.
The alkali-induced blue ¯uorescence of guard cells was
bright enough to distinguish them from the surrounding
epidermal cells. Thus the above procedure could be used as
a rapid and convenient method for in situ measurements of
the number, distribution and size of stomatal complexes.
Three main techniques have been established to quantify
the size of stomatal complexes and their frequency on leaf
surfaces (Willmer, 1983; Weyers and Meidner, 1990; Weyers
and Lawson, 1997). (1) Light microscopy of epidermal
strips. This is a convenient and low cost technique (Weyers
0
50
100
150
200
250
Image pixel intensity (grey scale)
F I G . 5. Sequence of changes in the intensity frequency plot (grey scale
values) resulting from the application of the ICF. A, Intensity
frequency plot of the (7,7) sub-image. Solid line: initial sub-image
frequency plot; dashed line: frequency plot after algorithm #1 of the
ICF; dotted line: frequency plot after algorithm #2 ( ®nal sub-image).
B, Intensity frequency plot of the whole image. Solid line: initial image
frequency plot; dotted line: frequency plot after ICF.
and Travis, 1981), however, the method is time consuming
and some distortion of the cells could cause alterations in
the epidermis, giving inaccurate measurements. Moreover,
epidermal strips are easily removed only in a limited
number of plant species. (2) Silicone rubber impressions of
the leaf surface. These provide a permanent record without
damaging the leaf, and their application in the ®eld is easy
(Sampson, 1961; Weyers and Johansen, 1985). However,
the procedure is relatively time-consuming. (3) Scanning
electron microscopy. SEM using ultra-rapid cryo®xation to
avoid aperture changes yields accurate measurements (Van
Gardingen et al., 1989). Nevertheless, the equipment
required is expensive and complex, and the capability for
observing large numbers of samples is limited.
The proposed experimental procedure includes a single
coating of a portion of the leaf surface by (or immersion of
the whole leaf in) a solution of 10 % KOH for 2 min,
washing with distilled water and direct observation under
the ¯uorescence microscope. Under the same experimental
Karabourniotis et al.ÐFluorescence Microscopy of Stomata
T A B L E 2. Stomatal aperture of leaves of V. faba plants
exposed to two di€erent irrigation treatments measured by
the proposed method (method A) and the replication method
(method B)
Stomatal aperture (mm)
Well watered leaves*
Water-stressed leaves
Method A
Method B
6.3 + 1.8
1.6 + 1.3
6.6 + 1.9
1.8 + 1.3
*See Materials and Methods. Data are means + s.d. from ®ve plants
for each treatment, three leaves per plant, three observations per leaf.
conditions the measurement of super®cial epidermal
appendices, such as trichomes, glands etc., would be
possible (Fig. 1B, see also image analysis section). The
suitability of the proposed method for measuring stomatal
apertures in species with relatively large stomata was tested
using V. faba plants di€ering in their water status. The
results obtained by the method described were comparable
to those obtained by taking leaf impressions of the same
leaves, either with closed or open stomata (Table 2). It was
also established that within the short time interval that
elapsed between coating with alkali solution and observation under the microscope, treatment with alkali did not
signi®cantly a€ect the stomatal aperture. However, K ‡
concentration and pH changes are involved in regulation
of the stomatal aperture; therefore the use of KOH at high
concentrations as a ¯uorescence inducer might alter the
aperture. For this reason, further experiments are needed to
ensure that the proposed method is suitable for measuring
stomatal pore aperture in other species.
Because either auto¯uorescence (in certain species) or
alkali-induced blue ¯uorescence of guard cells was bright
enough to provide sucient contrast to distinguish between
stomata (objects) and epidermal cells (background), it is
possible to count stomata by digital image analysis. It may
be possible to count objects on leaf surfaces using standard
image analysis techniques (i.e. directly converting the image
to binary and counting the objects). However each
application must be adapted to ®t the individual characteristics of each species.
To develop a computerized method for stomatal counting using the above technique, we applied the present digital
637
image analysis procedure. As a model, we chose a relatively
complex sample (abaxial surface of Olea europaea leaves,
see above). The image of this surface is characterized by the
occurrence of both ¯uorescing stomatal complexes and
trichome bases (Figs 1A and 6A). Additionally, epidermal
cells introduce noise that interferes in the process, the
background and the stomata/trichome bases show nonuniform intensity, and a major portion of the image may be
unfocused (Fig. 6A). Many of these disadvantages cannot
be reduced by more careful specimen preparation or
microscope use, because of the technical limitations of the
¯uorescence microscope and irregularities of the specimen
surface. For these reasons, the initial images were inappropriate for direct image analysis.
The process of image analysis prior to counting included
the application of the illumination correction ®lter (see
Materials and Methods). A representative image showing a
non-uniformly illuminated ®eld and unfocused areas is
shown in Fig. 6A. The application of the ICF (Fig. 6B)
produced an output image that showed uniform illumination in both the background and the image objects (stomata
and trichome bases). The intensity frequency plot of the
ICF-corrected image did not shift signi®cantly from the
initial position, however the frequency plot of the ICFcorrected image appeared smoother than the initial frequency plot due to intensity balancing (Fig. 5B). Application of median and background ®lters in the ICFcorrected image was e€ective in smoothing the image and
reducing the di€erence between well-focused and unfocused
objects. This allowed satisfactory division of the image into
objects, which were classi®ed as stomata or trichome bases
(Fig. 6C).
The deviation between results obtained by the computer
or from the human counting (which was considered to be
correct) was 4.9 % in the case of stomata, based on the
absolute di€erence values, and 1.0 % based on the algebraic
mean, while the deviation for trichome bases was 16.1 and
ÿ6 % respectively (Table 3). The low value of the algebraic
mean deviation indicates that the sum of deviations from a
series of counts is balanced around zero. The application of
the method, appropriately adapted (see Materials and
Methods) to another sample (abaxial surface of Vicia faba
leaves) showed similar results. These are considered to be
suciently accurate to provide a practical method for
counting stomata (Table 3). The blue channel image (see
T A B L E 3. Comparison between the human count and the image analysis count of the number of stomata and trichome bases
from the abaxial surface of dehaired olive leaves, and the number of stomata from the abaxial surface of faba bean leaves
(means + s.e.)
Olea europaea
Stomatal number per mm2 of leaf surface area
Number of trichome bases per mm2 of leaf surface area
Vicia faba
Stomatal number per mm2 of leaf surface area
Mean of human counts
Mean of computer counts
Mean of di€erence %
438.0 + 16.7
133.0 + 8.0
443.5 + 21.7
125.0 + 12.4
4.9 (1.0)
16.1 (ÿ6.0)
66.2 + 3.2
66.9 + 3.2
5.0 (1.0)
Di€erence % denotes the deviation of the computer count from the human count as the mean of the absolute values of the di€erences for each
individual sample (ten samples per plant). Values in parentheses are the algebraic means of the di€erences for each individual sample.
638
Karabourniotis et al.ÐFluorescence Microscopy of Stomata
overall process from the point of image acquisition until the
®nal count takes less than 2 min per image processed.
Our method could be extended to monitor in planta
changes in stomatal aperture, similar the other methods
using transmitted or re¯ected light (Omasa et al., 1983;
Omasa and Onoe, 1984; Kappen et al., 1987). However, at
present, this application could be used only in leaves whose
guard cells emit blue auto¯uorescence, as in the case of
olive leaves. Quantifying stomatal apertures will be the
subject of a future investigation.
AC K N OW L E D G E M E N T S
We thank Assistant Professor C. Fasseas for use of the video
camera, and Assistant Professor G. Theodoropoulos and the
Computer Centre of the National Technical University of
Athens for the use of computer programs.
L I T E R AT U R E C I T E D
F I G . 6. Abaxial surface of dehaired leaf of O. europaea. A, Initial
¯uorescence micrograph converted to 8-bit grey scale image. B, The
same image after the application of the ICF. C, The same image after
the overall image analysis sequence. Background is shown in black,
objects classi®ed as stomata are shown in red and objects classi®ed as
trichome bases are shown in white. Bar ˆ 100 mm.
Materials and Methods) was more sensitive in distinguishing
stomatal complexes from noise in that species. The method
is rapid: in routine work using an adequate PC system, the
Anonynous. 1998. Image-pro plus reference guide. Silver Spring, MD,
USA: Media Cybernetics.
Barnes JD, Cardoso-Vilhena J. 1996. Interactions between electromagnetic radiation and the plant cuticle. In: Kerstiens G, ed. Plant
cuticles. Oxford: Bios Scienti®c Publishers, 157±174.
Blanke MM, Pring RG, Baker EA. 1999. Structure and elemental
composition of grape berry stomata. Journal of Plant Physiology
154: 477±481.
Bohlar-Nordenkampf HR, Draxler G. 1993. Functional leaf anatomy.
In: Hall DO, Schurlock JMO, Bolhar-Nordenkampf HR,
Leegood RC, Long SP, eds. Photosynthesis and production in a
changing environment. London: Chapman and Hall, 91±112.
Fahn A. 1990. Plant anatomy. Oxford: Pergamon Press.
Harborne JB. 1989. General procedures and measurement of total
phenolics. In: Dey PM, Harborne JB, eds. Methods in plant
biochemistry. Vol. 1 plant phenolics. London: Academic Press,
1±28.
Harris PJ, Hartley RD. 1976. Detection of bound ferulic acid in cell
walls of the Graminae by ultraviolet ¯uorescence microscopy.
Nature 259: 508±510.
Johansen DA. 1940. Plant microtechnique. New York: McGraw-Hill.
Juniper BE, Je€rey CE. 1983. Plant surfaces. London: Edward Arnold.
Kappen L, Adresen G, Loesch R. 1987. In situ observations of stomatal
movements. Journal of Experimental Botany 38: 126±141.
Karabourniotis G. 1998. Light-guiding function of foliar sclereids in the
evergreen sclerophyll Phillyrea latifolia: A quantitative approach.
Journal of Experimental Botany 49: 739±746.
Karabourniotis G, Fasseas C. 1996. The dense indumentum with its
polyphenol content may replace the protective role of the
epidermis in some young xeromorphic leaves. Canadian Journal
of Botany 74: 347±351.
Karabourniotis G, Ko®dis G, Fasseas C, Liakoura V, Drossopoulos I.
1998. Polyphenol deposition in leaf hairs of Olea europaea
(Oleaceae) and Quercus ilex (Fagaceae). American Journal of
Botany 85: 1007±1012.
Kolattukudy PE. 1980. Biopolyester membranes of plants: Cutin and
suberin. Science 208: 990±999.
Liakopoulos G, Stavrianakou S, Karabourniotis G. 2001. Analysis of
epicuticular phenolics of Prunus persica and Olea europaea leaves:
Evidence on the chemical origin of the UV-induced blue
¯uorescence of stomata. Annals of Botany. doi:10.1006/anbo.1387.
Lichtenthaler HK, Schweiger J. 1998. Cell wall bound ferulic acid, the
major substance of the blue-green ¯uorescence emission of plants.
Journal of Plant Physiology 152: 272±282.
Morales F, Cerovic ZG, Moya I. 1996. Time-resolved blue green
¯uorescence of sugar beet (Beta vulgaris L.) leaves. Spectroscopic
evidence for the presence of ferulic acid as the main ¯uorophore of
the epidermis. Biochemica et Biophysica Acta 1273: 251±262.
Karabourniotis et al.ÐFluorescence Microscopy of Stomata
Omasa K, Onoe M. 1984. Measurement of stomatal aperture by digital
image processing. Plant and Cell Physiology 25: 1379±1388.
Omasa K, Hashimoto Y, Aiga I. 1983. Observation of stomatal
movements of intact plants using an image instrumentation system
with a light microscope. Plant and Cell Physiology 24: 281±288.
Palevitz BA, O'Kane DJ, Kobres RE, Raikhel NV. 1981. The vacuole
system in stomatal cells of Allium. Vacuole movements and
changes in morphology in di€erent cells as revealed by epi¯uorescence, video and electron microscopy. Protoplasma 109: 23±55.
Riley RG, Kolattukudy PE. 1975. Evidence for covalently attached pcoumaric acid and ferulic acid in cutins and suberins. Plant
Physiology 56: 650±654.
Rost FWD. 1995. Fluorescence microscopy. Vol. II. New York:
Cambridge University Press.
Russ JC. 1999. The image processing handbook. Boca Raton: CRC
Press.
Sampson J. 1961. A method of replicating dry or moist surfaces for
examination by light microscopy. Nature 191: 932±933.
Schnabl H, Weissenboeck G, Scharf H. 1986. In vivo-microspectrophotometric characterization of ¯avonol glycosides in Vicia faba
guard and epidermal cells. Journal of Experimental Botany 37:
61±72.
639
Van Gardingen PR, Je€rey CE, Grace J. 1989. Variation in stomatal
aperture in leaves of Avena fatua L. observed by low temperature
scanning electron microscopy. Plant, Cell and Environment 12:
887±888.
Weissenboeck G, Schnabl H, Scharf H, Sachs G. 1987. On the
properties of ¯uorescencing compounds in guard and epidermal
cells of Allium cepa L. Planta 171: 88±95.
Weyers JDB, Johansen LG. 1985. Accurate estimation of stomatal
aperture from silicone rubber impressions. New Phytologist 101:
109±115.
Weyers JDB, Lawson T. 1997. Heterogeneity in stomatal characteristics. Advances in Botanical Research 26: 317±352.
Weyers JDB, Meidner H. 1990. Methods in stomatal research. Essex:
Logman Scienti®c and Technical.
Weyers JDB, Travis AJ. 1981. Selection and preparation of leaf
epidermis for experiments on stomatal physiology. Journal of
Experimental Botany 32: 837±851.
Willmer CM. 1983. Stomata. New York: Longman.
Wollenweber E. 1985. Exudate ¯avonoids in higher plants of arid
regions. Plant Systematics and Evolution 150: 83±88.
Zeiger E, Hepler PK. 1979. Blue light-induced, intrinsic vacuolar
¯uorescence in onion guard cells. Journal of Cell Science 37: 1±10.