International Journal of Applied Engineering Research ISSN 0973-4562 Volume 11, Number 8 (2016) pp 5822-5825
© Research India Publications. http://www.ripublication.com
Optical detection technique for physical disorders in Capsicum annum leaves
Rakesh Kumar Jha, Mansik Jeon*, and Jeehyun Kim
School of Electronics Engineering, College of IT Engineering,
Kyungpook National University, 80, Daehak-ro, Buk-gu, Daegu 41566, Korea
noninvasively to measure scattering and absorption spectra of
apple skin as well as spectral shape of chlorophyll [ 10].
Another application of spectroscopic method includes
measurement of sugar content in melons [11]. Overall, the
above-mentioned modalities are inadequate in performing
depth-resolved, nondestructive imaging at the cellular level.
Abstract
Optical coherence tomography (OCT) was employed to reveal
physical disorders in Capsicum annum (pepper) leaves. Healthy
as well as abnormal Capsicum annum leaf samples with
various leaf spots were chosen for the experiment. The
acquired OCT cross-sectional and volumetric images
demonstrated several distinguishing attributes in subsurface
boundary regions of the leaf samples including individual leaf
layers and specific leaf d isorders. A-scan analysis further
clarified significant differences in the microstructures of leaf
sub-surface layers. The obtained results confirm OCT as a
noninvasive and nondestructive technique to reveal surface
structures and sub-surface cellular layers with high resolution
in Capsicum annum leaf diagnostics.
We demonstrate in this study the consequences of imaging
botanical samples with the implementation of optical
coherence tomography (OCT). The ultrahigh resolution
imaging capabilities of OCT can provide diagnostic
information on tissue microstructure that cannot be obtained
using other imaging modalities [12]. OCT uses low-coherence
infrared light to generate two- or three-dimensional crosssectional images of samples with micrometer resolution. OCT
is a rapid, non-invasive, interferometric imaging method used
for real time imaging. OCT has the advantages of minimal
sample preparation in comparison to conventional optical
methods and it enables the potential for periodic assessments
of the same sample over long duration. The OCT system,
which was initially developed by Fujimoto group for
ophthalmic application, has now widely been used in several
other research fields [13]. Moreover, OCT technology is
helpful for botanical research as it has appropriate scanning
depth range to explore the internal structure of leaves [14-19].
Keywords: Optical coherence tomography, Capsicum ann um,
Leaf spots, A-scan analysis.
Introduction
Leaf spots, which can be identified as circular lesions on
pepper leaves and stem, are highly destructive and can lead to
deformation of p epper leaves and fruits. Restoration of the
affected sample to its original state is quite unlikely. However,
several measures can be taken to prevent further expansion of
these spots. There are various germs that act as carriers of leaf
spots. Leaf spots mainly flourish in warm and heavy r ainfallprone areas. Plant rubble circulates the germs in the clay and
by means of infected seeds [1]. No pepper variety is immune
to leaf spots; however, leaf spots can be prevented by planting
spot-resistant varieties. Normally, it is too late to save the
pepper plants after appearance of any leaf spots on them [2].
Hence, development of a fast and nondestructive method for
the diagnosis of leaf spots is necessary.
A number of methods have b een executed for imaging
botanical samples, with special prominence on physiological
research and morphological imaging. Microscopic imaging
tools such as electron and light microscopes have been used
for in-depth analysis of cellular and morphological features,
which include extended sample preparation with actions like
sectioning, dehydration, and staining, forcing alteration in tissue
structure and are definitely destructive [3, 4]. Histological
operations can be exceptionally inconvenient in samples with
excessive water content, such as grapes [ 5]. Non-invasive
methods such as X-ray imaging and computed tomography
[6-9] and nuclear magnetic resonance imaging [5, 7] can be
applied for imaging entire samples; however, they possess
comparatively low resolution and are normally inefficient in
detecting cellular-level features. These imaging methodologies
for fieldare highly expensive and usually u nsuitable
implementation. Optical spectroscopic technique such as timeresolved reflectance of laser pulses has been used
The first dedicated report published on OCT applications for
botanical subjects described optical coherence microscopy
(OCM) as a technology for a rapid, in vivo, and nondestructive visualization of p lants and plant cells [20]. The
operating principle of OCM is almost similar with that of OCT
except that it uses more complicated imaging optics with high
numerical aperture to generate higher resolution enface
images. This system successfully performed in vivo monitoring
of cellular structures of rapidly growing plant leaves. To date,
several scientific reports on OCT application for plant tissue
have been published. Major research works include: study of
dissimilarities in the hull thickness in four distinct species of
lupine seeds [21]; detection of disease in melon seeds [22]
and apple leaves [23] for automatic screening of viral
infection in seeds and leaves, respectively. Reports on in-depth
study of detection of defects, rots, and diseases in onions were
also published [16, 24].
The objective of this study was to visualize and detect the
variations in the sub-surface cellular structures of healthy and
infected Capsicum annum leaves non-destructively, and
determine if differences which may be influenced by the
physical disorders were detectable. OCT imaging technology
was implemented to produce 2D and 3D images of the layers
of leaf structures, allowing subsequent analysis on the
microstructures of the Capsicum annum leaf structures.
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International Journal of Applied Engineering Research ISSN 0973-4562 Volume 11, Number 8 (2016) pp 5822-5825
© Research India Publications. http://www.ripublication.com
A-scan analyses were further performed for both the healthy
and abnormal leaf samples. The combined results were able to
justify the dissimilarities between healthy and abnormal
leaves.
compared with those of healthy leaf. All these experiments
were performed within few hours after collection of samples
to minimize the possible leaf-tissue damage with the passage
of time.
Materials and Methods
SD-OCT system configuration
Figure 1 demonstrates the Spectral-domain OCT (SD-OCT)
system configuration for 1310 nm. Imaging for healthy and
infected leaves was performed using our developed OCT
system. An infrared light source; super-luminescent diode
(EXS210046-01, Exalos, Switzerland) centered at 1310 nm with
full width at half maximum (FWHM) of 80 nm, is used in this
OCT system. A 5 0:50 fiber coupler was used to divide the
source power to the reference and the sample arms of the
interferometer, equally. In this system configuration, 2D
galvanometer scanner (GVS102, Thorlabs, USA) was used for
scanning the samples. The scanning range for B-scan images
was 2 mm.
Results and Discussion
The OCT imaging method, applied to a plant material, allows
visualization of the architectonics of surface-located and
underlying tissue layers. The acquired OCT images of the
Capsicum annum leaves, both with and without lesions,
demonstrate the capacity o f OCT to clearly identify the tissue
structures located at a depth of 1.5-2 mm. Figure 2 represents
the leaf structural images of healthy and abnormal Capsicum
annum leaves as visualized by OCT system. From the two
dimensional cross-sectional images obtained from the
experiment, the internal structures of the infected part of the
normal leaf were found to have possessed alterations after
being affected by the germs. Two dimensional cross-sectional
images were acquired for the selected regions shown in
Figures 2 (a) and (c), showing the upper epidermis, vascular
bundle, and mesophyll cells quite clearly, for the healthy leaf.
The abnormal leaf could not provide much information about
all these internal features of leaf, especially vascular bundles,
as the healthy regions of the abnormal leaf are also affected to
some extent by the lesions in the abnormal leaf. In particular,
the information revealed by this OCT system for the epidermal
cells and mesophyll cells is readily discernible. Three
dimensional volumetric scanning was performed for b oth the
healthy and abnormal leaves. Three dimensional images,
acquired by scanning the selected areas of the lesion in
abnormal leaf, were able to disclose the impact of the germ on
the lesion region. A-scan analysis was performed to obtain the
comprehensive details of the impact of the germ on the leaf.
The detection part of the OCT system contains a transmissiontype diffraction grating (HD 1145 l/mm, Wasatch Photonics,
USA), achromatic doublet lens (AC508-075-C, Thorlabs, USA),
and 1024-pixel line-scan InGaAs camera (1024-LDH2 92 KHz,
Sensors Unlimited, USA). By using X and Y-axis galvanometer
scanners, 500 B-scans images were acquired. A-scan analysis
was done to differentiate the layers of samples at micrometer
level. All A-scan profiles were normalized. The axial and
lateral resolutions for the system were measured in air as 6 µm
and 13 µm, respectively.
Figure 1: Schematic d iagram of spectral domain optical
coherence tomography (SD-OCT) system. Abbreviations: CL,
collimating lens; DG, diffraction grating; FC, fiber coupler;
GS, galvanometer scanner; L, lens; LSC, line-scan camera; M,
mirror; PC, polarization controller.
Plant sample preparation
Samples of healthy and abnormal Capsicum annum leaves
were selected for the experiment. After the collection of the
samples, the area to be scanned in the healthy leaf and that in
the abnormal leaf was determined as shown in Figures 2 (a)
and (c). The leaf spots on Capsicum annum leaves were
examined by differentiating lesion and healthy portions of the
infected leaf. The OCT images of the infected leaf were
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Figure 2: Visualization of Capsicum annum leaf structures
through OCT system. (a) normal leaf from a healthy tree; (b)
OCT image of healthy leaf; (c) abnormal leaf from a diseased
tree; (d) OCT image of abnormal leaf. Abbreviations: UE, upper
epidermis; MC, mesophyll cell; VB, vascular bundle.
International Journal of Applied Engineering Research ISSN 0973-4562 Volume 11, Number 8 (2016) pp 5822-5825
© Research India Publications. http://www.ripublication.com
Figures 3 represent the cross-sectional OCT images, A-scan
profile plots, and 3D OCT images of healthy and abnormal
Capsicum annum leaf samples. The A-scan profile depicted in
Figure 3(b) provides detail information about the internal
layers of the healthy leaf and that in Figure 3(e) gives detail
information about the healthy and defected regions of the
abnormal leaf. The X-axis and Y- axis represent the thickness
and the normalized intensity of the OCT signal, respectively.
The first peak that represents the up per epidermis (UE) layer
was found to be the same for both the healthy and abnormal
leaves. Furthermore, no other peaks were available for the
defected region of the abnormal leaf. However, the 2 and the
3 peaks were obtained in the healthy leaf and in the healthy
region of the abnormal leaf. Hence, as it can be observed from
the A -scan profile, OCT clearly reveals internal layerinformation of the healthy leaf, and the healthy region of the
abnormal leaf. However, due to leaf spots, the internal layers
of the defected region of abnormal leaf are merged together.
Similarly, A-scan profile p lots of the healthy and defected
regions show the distinguishable internal structures. In defected
region, no d istinct layers can be o bserved due to merging of
layers.
destruction of cellular components in the internal layers of
infected pepper leaf by g erms. Optical characteristics, such as
scattering coefficient of the leaf can play an important role in
providing a significant measure of the degradation of the leaf.
The alteration in the refractive indices of the cellular
components of the leaf tissue can also play critical role in
scattering at tissue layer borders. To sum up, this study strongly
suggests the usefulness of SD-OCT in botanical research for
analyzing the internal structural morphology, as the OCT
modality is rapid, non-invasive, high resolution technique, and
can be used for qualitative as well as quantitative analyses with
real-time image acquisition.
nd
rd
Acknowledgement
This research was s upported by Kyungpook National University
Research Fund, 2015.
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In conclusion, the application of OCT for the characterization
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© Research India Publications. http://www.ripublication.com
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