MEASUREMENT OF LEAF AREA WITH A HAND-SCANNER by cuiliqing

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									R. Bras. Fisiol. Veg. 4(1): 17-20, 1992



                      MEASUREMENT OF LEAF AREA WITH A
                   HAND-SCANNER LINKED TO A MICROCOMPUTERl

LINDA STYER CALDAS2, CARLOS BRAVO3, HOMERO PICCOLO3 and
CARMEN RACHEL S. M. FARIA2



Depto de Botânica, Instituto de Ciências Biológicas, Universidade de Brasilia,
CP 04631, Brasília, DF, CEP 70919, Brazil.


ABSTRACT- A method was developed for measuring leaf areas with                        Termos adicionais para indexação: análise de          crescimento,
a high degree of precision and ease of operation, using an inexpensive                herbivoria, fotossíntese, planímetro.
hand scanner coupled to an IBM-PC compatible microcomputer. The
software program, Area II, was developed by one of us (CB) and is
based on pixel counts of the images generated by the scanner, with
                                                                                                              INTRODUCTION
an appropriate calibration factor. A set of 18 leaves of two species of
Eucalyptus was photocopied and the areas determined by planimeter,                       Leaf area must be determined for many physiological studies,
gravimetrically (weighing the paper copy of the leaf and comparing the                such as growth analysis, photosynthesis and transpiration mea-
weight with known areas of the same paper) and with a leaf area meter,                surements. Reviews of the techniques normally used for the
as well as the scanner. Each leaf was measured four times by each                     determination of leaf area (e.g. Kvêt & Marshall, 1971; Coombs
method and the coefficients of variation of the areas were calculated                 et al, 1985) cite the leaf area meter as the usual method, although
for each method. The coefficients of variation for the leaves of the two
                                                                                      planimeters, photogravimetric methods and area-length regres-
species of Eucalyptus were of the same order of magnitude as those
obtained with the leaf area meter, and smaller than those obtained with               sions are also mentioned by these authors.
the gravimetric method and the planimeter. Extremely small leaf areas                    A comparison of these methods for the determination of a large
(less than 0.15 cm2) can be measured by using the high resolution (400                number of leaf areas shows that the area-length regressions must
dpi) adjustment on the scanner. The method is useful for growth analysis,             be performed for each species, and even for different treatments
photosynthesis measurements and studies of herbivory, since the                       within a certain species. For this regression, the area must be
damaged areas can be easily completed using the software functions                    independently determined, using one of the other methods.
after the image has been generated.                                                      Although the gravimetric method based on the weight of the paper
Additional index terms: Growth analysis, herbivory, photosynthesis,                   cut-out of the leaf tracing (or photogravimetric method,
planimeter.                                                                           when photocopies of the leaves are used), compared to the
                                                                                      weight of known areas on the same paper, provides an accurate
MEDIÇÕES DE ÁREA FOLIAR COM UM “HAND-SCANNER”                                         measurement of the area, it is a laborious technique when applied
ACOPLADO A MICROCOMPUTADOR                                                            to a large number of leaves.
                                                                                         The planimeter offers a less time-consuming technique, but the
RESUMO- Um método foi desenvolvido para medir área foliar com                         precision is limited, especially for relatively small leaves.
um elevado grau de precisão e facilidade de uso, com base num “hand                      Leaf area meters have been widely adopted for these mea-
scanner” de baixo custo acoplado a um microcomputador compatível                      surements because of their ease of operation. Experience has
com o padrão IBM-PC. O programa, Area II, foi desenvolvido por um
                                                                                      shown that the other methods are tedious, particularly when a
dos autores (CB) e funciona, com um fator de calibração apropriado,
na base da contagem dos pixels pretos em imagens geradas pelo                         large number of leaves must be measured.
“scanner”. Foram feitas fotocópias de um conjunto de 18 folhas de duas                   More recently, methods involving image processing based on
espécies de Eucalyptus e as áreas medidas por planimetro, pelo peso                   video camera images and computer programs for analysis of
da fotocópia da folha recortada e por um medidor de área foliar, além                 these images, have been proposed, for example, Hargrove &
do “scanner”. Para cada método, a área de cada folha foi medida quatro                Crossley, 1988; Hagerup et al, 1990).        These methods permit
vezes e os coeficientes de variação das medidas calculadas. Os                        automatic calculation of leaf areas, and of areas lost to herbivores
coeficientes de variação para as folhas das duas espécies de Euca-                    or disease, depending on the computer programs used.
lyptus foram da mesma ordem de magnitude com o “scanner” e com
o medidor de área foliar e inferiores aos valores obtidos pelos métodos
                                                                                         The following method is presented as a less expensive option
gravime’trico e planimétrico. Folhas extremamente pequenas (menores                   to the leaf area meter and the video camera image processing.
que 0,15 cm2) podem ser medidas utilizando o ajuste de alta densidade                 The alternative method uses a hand scanner linked to an IBM-
(400 dpi) do “scanner”. 0 método é útil para análise de crescimento,                  PC compatible microcomputer. The image of the leaf generated
medidas de fotossíntese e transpiração e de herbivoria, pois as áreas                 by the scanner is transformed to area by an appropriate software
danificadas podem ser facilmente completadas com as opções do                         program, which is based on the pixel counts of the image.
software após a geração da imagem.
                                                                                                          MATERIAL AND METHODS
1
  Recebido em 20/03/92 e aceito em 04/06/92.
2
  Prof. Adjunto                                                                       Description of the Scanner Method
3
 Aluno de graduação, Depto de Ciências da Computação, Universidade de Brasília,
CP 4640, Brasília, DF, CEP 70919.                                                        The computer program, Area II, was developed by one of us
4
 Prof. Assistente, Depto de Ciências da Computação, Universidade de Brasília,         (CB) and is based on a pixel count of the image generated by
CP 4640, Brasília, DF, CEP 70919.                                                     a hand scanner. A GeniScan TM GS-4500 hand scanner was used
5
 Prof. Auxiliar Visitante, Depto de Botânica, CP 04631, Brasília, DF, CEP 70919.

                                                                R. Bras. Fisiol. Veg. 4(1):17-20, 1992.
18



in the present tests, with ScanEdit II TM software. The scanner                uniformly black with an appropriate adjustment of the light-dark
has settings for letters (two-tone or black-and-white) or photo-               setting of the scanner during image generation.
graphs (32 levels of “gray scale”), 100 to 400 dpi resolution and                  Initial tests before saving the image allowed the adjustment of
a variable light-dark adjustment to control the intensity of color             the setting. Eucalyptus torelliana leaves, for instance, gave sharply
in the image generated. The maximum image width is 105 mm                      contrasted, homogeneous images from both the dark green,
for a single scan but images up to 300 mm wide can be incor-                   adaxial surface and the lighter green, abaxial surface. Yellow,
porated with the merge option. The equipment can be used with                  senescent leaves of Catharanthus gave very satisfactory images
an IBM-PC compatible microcomputer with a minimum of 640 kB                    against the same white paper background, with a darker adjust-
memory and a CGA video monitor.                                                ment.
    Detached fresh leaves with petioles removed were flattened                     Necrotic lesions of leaves of Catharanthus and leaflets of woody
against a white or light-colored background and covered with a                 leguminous species ( Dalbergia miscolobium and Pterodon
sheet of stiff, transparent plastic before passing the hand scanner            pubescens) appeared as white areas in the images generated
with appropriate adjustment of the “Light-Dark” control. The                   by the scanner. Video camera images were analyzed by Lindow
scanner was always used with the letter (black-and-white) setting              & Webb (1983) to detect the area affected by necrosis, but their
and 100 dpi for larger leaves (above O.l5 cm2). Very small leaves              system required a black velvet back-ground and red light illumi-
(less than 0.15 cm2) from tissue culture potato plants, courtesy               nation to maximize contrast. The yellow-green, built-in light source
of Lu’cio Fla’vio de Alencar Figueiredo and Eliseu Soares Figueira             of the hand scanner gave sufficient contrast for several types of
Filho (CENARGEN-EMBRAPA, Brasi’lia, DF), were measured with                    necrotic lesions to be distinguished in the present, simple system.
200 and 400 dpi settings as well.                                                  In the case of photocopies, a sheet of millimeter scaled graph
    Images were also generated from photocopies of leaves when                 paper was copied four times to detect possible errors in area
a large number of measurements were made. Tracings of the                      due to reduction or amplification of the leaves by the copier; an
outline of leaves which could not be detached are also suitable                increase of 1% in the area of the image occurred with the copying
for use with this program. In these cases, the image generated                 machine we used.
by the scanner is composed of the outline of the leaf and the                      The tracings of small leaves, or photocopies with only the
“Fill” function in the Graphic Tools of the software can be used               border of the leaf appearing, can easily be filled with the “Fill”
to easily fill the image with solid black color before saving and              function of the scanner software. With the leaf tracings or pho-
analyzing the area.                                                            tocopies which require filling, it may be necessary to complete
    The Area II program analyzes the image saved with default                  the borderline of the leaf before filling to avoid the “overflow” of
extension, .TIF, of ScanEdit II. Each leaf is saved as a single, sub-          the fill pattern to the area through a break in the outline. This is
image file since Area II will provide the total area of all black              most easily done by retouching the outline before scanning, in
images in the file. The Area II program offers the option of analyzing         the points where the line is faint or broken. Nolting & Edwards
a series of files and transferring the areas to another file (TEST.DOC,        (1985) used a light pen with their video camera system to com-
for example). All areas read by Area II will be appended to the                plete the margins of partially defoliated leaves prior to estimation
TEST.DOC file when this command is used, without erasing                       of total. undamaged leaf area.
previous registers.                                                                The images of larger leaves do not fit on the CGA screen used
    The program was calibrated by generating images with a series              in the present system, which makes filling a more laborious task.
of squares and rectangles cut from graph paper with millimeter                 The image must be called up in several parts for filling before
divisions. Areas from 0.01 to 108 cm2 were used for the calibration            saving for area analysis. If possible, these leaves, or the photo-
with at least five independent images for each area. Blank images              copied images, can be cut into smaller pieces and the area
always gave an area of 0.00 cm2.                                               obtained as the sum of the partial images. This method is pref-
                                                                               erable to the use of the “Merge” function of the scanner software.
Comparison with Other Methods                                                      Multiple images can be saved in a single file, when the leaf is
                                                                               undulating and must be cut into several pieces to lie flat for
    Sets of nine leaves of Eucalyptus citriodora and nine leaves               scanning. The program will give the total area of all images in
of E. torelliana were photocopied and used in all the subsequent               a file, based on a total black pixel count. This type of analysis,
measurements. One copy was used for the planimeter measure-                    although it involves saving each leaf as a single file, makes the
ments, which were repeated four times on each leaf image with                  calculation of the leaf areas very rapid (approximately 1 second
an Allbrit planimeter.                                                         for leaf areas of 50 cm2), when compared with the method of
    Four copies were used for cutting out the leaf images and                  Hagerup et al (1990), in which the images of several leaf discs
weighing them on a Mettler H35 analytic balance, with a sensitivity            are stored in a single file and the algorithm must discover and
of 0.1 mg. Thus four separate weighings on the four copies were                calculate the areas of separate leaf discs within the file, or picture
obtained for each leaf. For calibration of the weight, squares of              (20 seconds to estimate the “initial” area without insect feeding
4 cm2 area were cut from the same sheet of paper and weighed                   and calculate the percentage of the area lost to feeding for each
to provide the specific calibration factor for each copy.                      disk).
    One copy of each leaf, previously used for the gravimetric                     In the present method, the “Fill” function of the ScanEdit soft-
determination, was measured four times in the Hayashi Denkoh                   ware was used to complete areas removed by insect herbivory,
Model AAM-7 Leaf Area Meter, after calibration of the instrument               affected by disease, or removed for measurements of enzyme
following manufacturer’s instructions.                                         activity. This was done after saving the image of the original,
    One copy of the set of leaves was used for scanner measure-                damaged leaf. A second image was generated and the difference
ments. Four images were generated for each leaf and the areas                  between the two areas used to calculate the percentage of leaf
determined with the Area II program.                                           area affected. This procedure is made easier by retouching the
    The average area and coefficient of variation for each leaf were           presumed outline of the undamaged leaf on a photocopy before
calculated for each method.                                                    the second scan, when the area affected by grazing is on the
                                                                               edge of the leaf.
              RESULTS AND DISCUSSION                                               Alternatively, the “Invert Image” command of the ScanEdit
                                                                               software can be used to transform the white areas of damage
     The images produced from fresh leaves were sharp and                      to black areas, for direct measurement of the damage, while the

                                                         R. Bras. Fisiol. Veg. 4(1):17-20, 1992.
                                                                                                                                                            19



rest of the leaf area becomes white.
   The calibration factor was calculated from a set of squares and              With the methods which use video cameras to generate the
rectangles of known area. Least-squares fit of the known areas               images, a separate calibration must be carried out for each distance
with the areas determined by the Area II program gave a cor-                 from the camera to the leaf (Lindow & Webb, 1983: Noting &
relation coefficient R2=1.0000; the equation relating the scanned            Edwards, 1985; Hargrove & Crossley, 1968). Since this distance
area to the known area was:                                                  is constant with the hand scanner, a single calibration factor can
                                                                             be used.
Scanned Area = 1.001*(Known Area) - 0.0621                                      The software program Area II was adjusted to give a resolution


TABLE 1- Leaf areas measured by four methods. The same leaves were measured four times each by each method.

  Leaf                                        Planimeter                      Gravimetric                        Leaf Area Meter                     Scanner

Eucalyptus                           Area             cv           Area                  cv              Area                 cv          Area            cv
 citriodora                          cm2              %            cm2                   %               cm2                  %           cm2             %
     1                                4.5           9.07           4.50               2.04                4.77              0.43           4.46         0.13
     2                                5.9          12.77           6.55               3.10                6.94              0.82           6.49         0.52
     3                                6.9          10.91            7.61              1.73                7.79              2.63           7.51         1.20
     4                               15.3           3.28          15.95               1.37               16.24              0.18          15.30         0.75
     5                               16.6           2.88          17.50               1.50               18.08              0.36          17.06         0.36
     6                               21.6           7.41          22.13               1.06               22.62              0.28          21.22         0.43
     7                               20.8           2.41          23.37               2.18               23.68              0.13          22.56         0.29
     8                               23.4           3.21          24.BB               2.16               25.20              0.32          23.72         0.27
     9                               24.8           1.17          25.32               3,17               25.89              0.37          24.82         0.23
Eucalyptus
 torelliana
     1                                3.0          13.61           3.70               5.85                4.0O              O.94            3.7l        1.33
     2                                3.3           8.88           4.09               3.35                4.29              0.77           3.97         1.72
     3                                7.5           5.44           8.34               2.26                8.84              0.72           8.31         1.00
     4                                7.6           6.28           9.77               1.30               10.29              0.63           9.67         0.45
     5                               14.5           2.82          15.05               1.51               15.70              O.38          14.89         O.26
     6                               37.4           4.00          37.36               0.77               38.49              0.20          36.51         0.23
     7                               42.3           2.82          43.50               2.08               44.82              O.05          42.78         0.23
     8                               49.5           1.43          49.69               1.66               50.90              0.22          48.64         0.17
     9                               49.4           0.00          49.93               0.00               51.67              0.00          49.43         0.00

of 0.01 cm2. There is no difference in the area given by the                 pixels. The Area II program will calculate the area based on the
software program for a single image, when the area is determined             calibration factor for 100 dpi. If the image was generated with
several times. Since there is no need to trace the outline of the            the 400 dpi setting, for instance, the given area must be divided
leaves or cut the paper form, a high degree of reproducibility is            by 16 to correct for the larger number of pixels. The coefficient
also possible with the scanner. This can be seen in Table 1, where           of variation for areas which appeared as 0.01 cm2 when analyzed
the coefficients of variation of the measurements of leaves of               at 100 dpi was 77% (Table 2). The same areas, measured at 400
Eucalyptus citriodora and E. torelliana using the scanner were               dpi, gave average areas of 0.014 and 0.015 cm2, with coefficients
comparable to those obtained with the leaf area meter. The                   of variation of 4.4 and 5.51% (Table 2). Areas in the range of O.1O
coefficients of variation with the scanner were one-half to one-             to 0.25 cm2 can be measured at 400 dpi with a coefficient of
tenth of those obtained with the gravimetric method and one-third            variation ranging from 0.5% to 2.3% (Table 2) while even smaller
to one-fiftieth of the coefficients obtained with the planimeter.            areas (from 0.015 cm2) were measured with a maximum coef-
   The area analysis system of Nolting and Edwards (1985) gave               ficient of variation of 11.6% (Table 2).
significantly lower standard deviations (P<0.05) for repeated
measurements of the same objects than the leaf area meter.                   TABLE 2- Minimal leaf areas measured at three resolutions. The same leaves were
   The average leaf areas obtained with the scanner for the smaller          seasured four times each at each resolution.
leaves are closer to the values from the gravimetric method than
                                                                                                     100 dpi                  200 dpi                  400 dpi
to the values from the planimeter or the leaf area meter. Linear
regression of the measurements of the different methods as a                                  Area       cv          Area           cv        Area         cv
function of the scanner areas showed the closest agreement                   Leaf             cm2        %           cm2            %         cm2          %
between the gravimetric method and the scanner (slope of 1.015),             1                0.01       77         0.012          20.2      0.014        4.4
while the planimeter and the leaf area meter gave slightly higher            2                0.01       77         0.009          28.6      0.015        5.5
values for the areas (slopes of 1.020 and 1.028, respectively).              3                O.02       38         0.013          36.1      0.016        6.8
                                                                             4                O.02       29         0.022          19.5      O.022       11.6
   Lindow & Webb (1983) showed a good correlation (coefficient               5                O.03       33         0.023          14.0      0.027        2.2
of determination = 0.99) between the areas of bean leaves                    6                0.03       50         0.028           8.9      0.032        9.0
measured with their digitized video image analysis and the areas             7                0.02       29         0.032           3.9      0.036        2.3
measured with a planimeter. They did not describe the calibration            8                0.03       31         0.035          10.1      0.041        4.0
procedure for the image analysis and used the planimeter                     9                0.02       22         0.036           8.0      0,041        3.1
measurements as the true area.                                               10               0.06       22         0.068           6.8      0.073        1.9
                                                                             11               0.10        8         0.108           3.3      0.111        0.5
   A high degree of precision can be achieved in measuring very              12               0.11        7         0.118           2.0      0.126        0.5
small leaf areas (less than 0.l5 cm2) by adjusting the scanner               13               0.12        8         0.133           4.6      0.145        1.2
to 200 or 400 dpi, instead of 100 dpi (Table 2). An area of 0.01             14               0.15        4         0.155           0.0      0.157        2.3
cm2 appears as 16 pixels after scanning at 100 dpi; at 200 dpi,              15               0.22        7         0.233           2.6      0.256        0.9
the same area gives an image with 64 pixels and, at 400 dpi, 256             16               0.24        3         0.243           4.6      0.264        1.3


                                                       R. Bras. Fisiol. Veg. 4(1):17-20, 1992.
20



    In comparison, the smallest area measured with the video                    Copies of the program are available from                        the authors.
camera/digitizer system of Lindow & Webb (1983) was 20 cm,
while Hargrove & Crossley (1988) indicated a minimum of 5 cm2                                     ACKNOWLEDGMENTS
and Hagerup et al (1990) measured leaf discs with an area of
1.65 cm2. Nolting & Edwards (1985) were able to measure objects                We thank Carlos Lazarini Fonseca, of CPAC/EMBRAPA, for his
with a minimum area of 0.25 cm2. The high degree of resolution              help with the Leaf Area Meter; Heloisa Sinatora Miranda and Ruy
of the present method makes it particularly suitable for the                de Araujo Caldas, for critical reading of the manuscript, and Kiniti
measurement of small leaf areas; it is more laborious when large            Kitayama, for providing useful bibliography.
leaves must be measured, frequently entailing a preparation
process to cut the leaves up into smaller parts, which will also                                         REFERENCES
lie flat, without undulations.
    In summary, the present method proved to be easy to use, in                                                         .
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                                                                            HAGERUP M.; SONDERGARD, I. & NIELSEN. J. K. Measurements of areas consu-
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software program and method can be applied to measure the                       Production. Manual of Methods. The Hague, Dr W. Junk. 1971. pp. 517-555.
area in closed figures on graphs or maps, for integrating daily             LINDOW, S.E. & WEBB, R.R. Quantification of foliar plant disease symptoms by
                                                                                microcomputer-digitized video image analysis. Phytopathology, 73:520-524,
totals of CO2 fixation, transpiration or many other applications.               1983.
This flexibility makes the program of general use, although                               .
                                                                            NOLTING, S.P & Edwards, C.R. Defoliation assessment using video imagery and
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                                                      R. Bras. Fisiol. Veg. 4(1):17-20, 1992.

								
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