Microstructure, Extinction Coefficient, and Chlorophyll Content of

Philippine Bamboo Leaves by a Portable TD-OCT Scanner

Jumar Cadondon

1,2,3 a

, Edgar Vallar

1 b

, Maria Cecilia Galvez

1 c

and Tatsuo Shiina

3 d

Department of Physics, College of Science, De La Salle University, 1004 Taft Avenue, Manila 0922, Philippines

Division of Physical Sciences and Mathematics, College of Arts and Sciences-Miagao Campus, Miagao 5023,

Iloilo, Philippines

Graduate School of Engineering, Chiba University, Yayoi-cho, Chiba 263-8522, Japan

Keywords: Philippine Bamboo, Extinction Coefficient, Microstructure, Chlorophyll, TD-OCT.

Abstract: Bamboo is one of the most utilized non-timber forest products in the Philippines. Common bamboo leaf

infections are caused by sunlight, and nutrient deficiency. In this study, we have developed a portable time

domain-optical coherence tomography (TD-OCT) to study in vivo leaf microstructure changes in Philippine

bamboo (Bambusa spinosa). TD-OCT analysis shows unique features among different layers of the leaves

specifically on the epidermis and palisade layers when the unhealthy part is compared to the healthy part.

Extinction coefficient from the A-scan analysis showed significant difference from unhealthy part (1.03 ±

0.20 mm

-1

, N =12, p<0.05) and healthy part (0.72 ± 0.27 mm

-1

, N =12, p<0.05). In addition, RGB data was

compared for both unhealthy and healthy part of the bamboo leaves. A red shift is observed from the unhealthy

part of the leaves. It is also observed by a decrease of 60% in chlorophyll-a content of the unhealthy part as

compared to the healthy part. Same inverse correlation is also observed when the extinction coefficient is

compared with the chlorophyll content.

1 INTRODUCTION

Bamboo is a diverse group of perennials with emerging

use in food, handicrafts, chemical products, and

building materials (Cheng et al., 2023). The

microstructure of leaves is crucial in the overall

development of plants. Leaf growth depends on its

photosynthetic ability and leaf phenology. Visual

inspection shows unhealthy and healthy structure

within the leaves. Furthermore, the loss of the

photosynthetic ability of the plant is due to

environmental factors such as temperature, humidity,

nutrition, and oxidative stress (Liu et al., 2022). Several

methods have been introduced in leaf growth dynamics

and early detection of leaf diseases. Such inspection are

subjective, inefficient, time-consuming methods in

early detection of leaf diseases. Understanding such

relation between chlorophyll concentration and its

microstructures has not been further studied.

https://orcid.org/0000-0002-3933-0598

https://orcid.org/0000-0001-8236-7102

https://orcid.org/0000-0001-5505-1778

https://orcid.org/0000-0001-9292-4523

Traditional methods using biomedical techniques

have been commonly used to study photosyntheitc

ability in plants such as chlrophyll content, protein,

lipids, and fats (Cao et al., 2013). However, these

studies mostly focus on the cell components. In vivo

technique using microstructural patterns have been a

growing research on the leaf plant development.

Changes in the leaf microstructures are mostly

associated with its photosynthetic ability. Imaging

techniques such as scanning electron microscopy

(SEM), transmission electron microscopy (TEM)

(Wang et al., 2014; Yao et al., 2017), confocal and

fluorescence microscopy (Zhao et al., 2016) and

spectroscopic methods (Butler et al., 2015; Ivanova

and Singh, 2003) can provided cellular, molecular

data, however, are limited by penetration depth which

requires plant sectioning.

Optical coherence tomography (OCT), is a non-

invasive technique that can provide high-speed cross-

114

Cadondon, J., Vallar, E., Galvez, M. C. and Shiina, T.

Microstructure, Extinction Coefﬁcient, and Chlorophyll Content of Philippine Bamboo Leaves by a Portable TD-OCT Scanner.

DOI: 10.5220/0013244900003902

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 13th International Conference on Photonics, Optics and Laser Technology (PHOTOPTICS 2025), pages 114-119

ISBN: 978-989-758-736-8; ISSN: 2184-4364

sectional imaging with micrometer resolution in

highly scattering samples. OCT has been widely used

for retinal imaging, dermatology, forensic studies

(Popescu, 2014; Meglinski et al., 2010). There are

increasing trends in using OCT in biomedical

applications in plant biology and agriculture (Goto et

al., 2023). In this study, we have developed a time

domain- OCT (TD-OCT) that shows deeper

penetration in leaves. In this study, TD-OCT has

been used to obtain cross-sectional images of

Bambusa sp. leaves to illustrate microstructural

differences in healthy and unhealthy part due to

environmental factors. Moreover, the extinction

coefficient measured from A-scan analysis was

compared for both healthy and unhealthy parts of the

bamboo leaves. The chlorophyll-a content was also

studied using absorbance spectroscopy. Correlation

between the exiction coefficient and chlrolophyll-a

content has been observed using the developed TD-

OCT. This is the first reported study on the use of TD-

OCT imaging in elucidating microstructural changes

in leaves which can be furthered explored for

agricultural growth and development.

2 TD-OCT DEVELOPMENT

Our TD-OCT is based on the Michelson

interferometer as shown in Fig 1. Our OCT has

dimensions of 10 in. by 8 in. 5 in., which can be easily

used for in-situ plant monitoring. A rotating

retroreflector is designed using a reference arm (RA)

instead of moving in translation motion. It has more

scanning range which can be easily adjusted by

changing the retroreflector’s radius. A 1310 nm SLD

(Anritsu Co. Ltd., Kanagawa, Japan) with a spectral

width of 56 nm and average axial resolution of 14.2

μm in air was used (Shiina et al., 2003). It is

constructed to evaluate the microstructural changes of

the plant by acquiring A-scans as point measurement.

Table 1 shows the specifications of the developed

TD-OCT (Galvez et al., 2023). In this system, the

probe is designed to be small and can be position

easily on the surface of the leaves.

It includes super luminescent diode (SLD),

photodiode (PD), signal processing circuit board

(SPCB), oscilloscope (Osc), personal computer (PC),

beam splitter (BS), reference arm (RA), probe (PR),

sample (SP), and fiber coupler assembly (FCA).

Figure 1: The schematic diagram of the TD-OCT system.

Table 1: Specifications of the TD-OCT system.

Specification

Value

Center wavelength

1310 nm

Spectral width

56 nm

Axial resolution

14.2 μm

Lateral resolution, spot size

6 μm

Numerical aperture

0.14

Scanning rate

25 scans/s

Scanning depth in air

12-14 mm

2.1 Bamboo Collection

Bamboo has disadvantages like pest and fungal

susceptibility due to its small morphological

structure. It has also low survival rate for

micropropagation. On the other hand, growing

bamboo has the ability to mitigate flood and soil

erosion (DOST PCAARD, n.d.). The OCT

measurement was conducted using Philippine

bamboo collected from the Philippines’ Department

of Agriculture. Twenty-five (25) bamboo leaves were

collected based on visual inspection showing both

healthy and unhealthy part (Fig. 2).

Figure 2: The bamboo leaf (top view) collected with the

healthy and unhealthy part.

Microstructure, Extinction Coefﬁcient, and Chlorophyll Content of Philippine Bamboo Leaves by a Portable TD-OCT Scanner

115

2.2 Microstructural Imaging, and

Extinction Coefficient

In this work, all bamboo leaves were imaged on

adaxial surface. The photographs (Fig. 2) emphasize

the topographical and color changes. Leaves are

multilayered structures which varies in absorption

coefficients in different layers. Hence, A-scan and B-

scan analysis was performed to obtain a detailed

microstructural information from the TD-OCT

images. A-scan is defined for depth scan which is also

related to longitudinal scan; while B-scan is referred

to the transverse sections. The thickness between two

layers of a leaf can be defined between corresponding

distance between the A-scan profile. With the

successive A-scan analysis, a 2D-cross sectional

image is created, called the B-scan.

Using the microstructural information collected,

these can be quantified in terms of intensity by

measuring the extinction coefficient. The extinction

coefficient is an intrinsic optical property of tissues

that is highly correlated with the TD-OCT signals. It

is defined as the amount of light scatter and

absorption per unit distance as light travels into the

plant tissue.

2.3 RGB Image and Chlorophyll-a

Measurement

An Olympus microscope with a high-resolution

camera attached was used to analyse the adaxial

information (Fig. 2) and RGB images. To measure

specific frequency distribution of the RGB intensity,

a region of interest (ROI) was chosen. This is also

designated as the region where OCT imaging was

conducted. In Fig. 2, the red rectangle represents the

ROI. The difference in the RGB frequency was

recorded for both healthy and unhealthy part of the

bamboo leaves.

Microstructural and morphological changes are

accompanied by the changes in the chlorophyll

content of the leaves. Thus, we also measured the

chlorophyll-a concentration by measuring the optical

density (OD) of the leaves at 680 nm (Cadondon et

al., 2023) and the estimated chlorophyll-a

concentration based on Sartory and Grobbelaar

(1984) as shown below.

Chl − a =

[

26.73

(

665a − 663b

)]

(1)

A detailed methodology is presented previously

(Cadondon et al., 2022). The optical density was

measured at 750 nm, 663 nm, and 630 nm

wavelengths. In the equation above, 665a is the

turbidity corrected absorbance at 665 nm, and 663b is

the turbidity corrected absorbance at 663 nm after

acidification, F is the dilution factor, E is the volume

of the solved used for the extraction (mL), V is the

volume of the filtered sample (mL), and L is the path

length (cm). Unsaturated samples are given with a

dilution factor of 1. On the other hand, dilution factor

of saturated samples is dependent on the number of

times the samples are diluted. In this case, the volume

of the solvent also increases.

2.4 Statistical Treatments

Using Pearson’s R correlation, the estimated

chlorophyll-a concentrations of the bamboo leaves

were correlated with the extinction coefficient

measured from the OCT signals.

3 MICROSTRUCTURE

ANALYSIS OF BAMBOO

LEAVES

Figure 3 (a) and (b) shows the microstructural

changes of the bamboo leaves on OCT images.

Corrections were made by subtracting the

background light, focal length correction, distance

squared correction, and logarithmic analysis to

obtained the B-scan signals. The broadening of the

second order peak in the unhealthy part of the leaves

is highly observed. This implies that the upper dermis

and palisade layers merged to form a thick layer. The

increase in the thickness and the changes in the

epidermis and palisade layers can be used to explain

the environmental factors affecting the color change

in the bamboo leaves. Hence, it is very useful to

identify microstructural changes on the leaves using

OCT images to further understand its morphological

changes.

This can be further verified by determining the

frequency distribution of the RGB color in the leaves.

Fig. 4 (a) and (b) provides the intensity-frequency

distribution of the RGB image of the healthy and

unhealthy part of the bamboo leaves. The x-axis is the

intensity and the y-axis is the frequency distribution

of the color.

PHOTOPTICS 2025 - 13th International Conference on Photonics, Optics and Laser Technology

116

(a) Healthy part

(b) Unhealthy part

Figure 3: The OCT images of the bamboo leaves collected

with the healthy and unhealthy part.

The same intensity and frequency distribution is

observed in a healthy part of the bamboo leaves as

shown in Fig. 4(a). A decrease in the intensity profile

is observed in the unhealthy part of the bamboo

leaves. A shift in the red profile is also observed (Fig

4(b) (Li et al., 2012). This means that the absorption

of the bamboo leaves changes as the microstructure

changes (Zhang et al., 2022).

(a) Healthy

(b) Unhealthy

Figure 4: Normalized Intensity-Frequency profile of the

RGB of healthy and unhealthy part of the bamboo leaves.

4 EXTINCTION COEFFICIENT,

AND CHLOROPHYLL

CONTENT

As discussed, extinction coefficient is important to

quantitatively measure the microstructural changes in

terms of intensity. Figure 5 shows the extinction

coefficient estimated by OCT images. From the A-

scan measured, a lineal fitting model was structured

to obtain the slope of the extinction coefficient of

each. The calculated mean extinction coefficients ±

standard deviation for healthy and unhealthy part are:

0.72 ± 0.27 mm

-1

, and 1.03 ± 0.23 mm

-1

, respectively.

Our results statistically show that lower extinction

coefficient is observed for the healthy part as

compared to the unhealthy part. This explains the

broadening of first layer and the second layer with the

merging of the epidermis and the palisade layer

(Anna et al., 2018). The microstructural changes in

the upper dermis provides information on the

adapting ability to environmental factors.

Microstructure, Extinction Coefﬁcient, and Chlorophyll Content of Philippine Bamboo Leaves by a Portable TD-OCT Scanner

117

Figure 5: Mean extinction coefficient of the bamboo leaves.

To confirm the changes in the intensity and

frequency distribution from the RGB data along with

the morphological and microstructural changes in the

bamboo leaves, the chlorophyll-a content is measured

(Fig. 6). A significant decreased by 60 % was

observed from the unhealthy part of the bamboo

leaves when compared with the healthy part. These

findings show that the chlorophyll-a content varies on

the different parts of the leaves. This also confirms

that such decrease in affected by environmental

factors that lowers the ability of the plant to produce

its own chlorophyll.

Figure 6: Mean chlorophyll-a content of the bamboo leaves.

With the significant change, it can be observed

that there is an inverse correlation between the

extinction coefficient of bamboo leaves based on the

OCT image and the chlorophyll-a content (Inskeep

and Bloom, 1985). Visual inspection through

microscopy and cell analysis are commonly used in

understanding chlorophyll-a distribution in plants. In

this study, we were able to provide a different

approach in estimating chlorophyll-a distribution

using the estimated extinction coefficient from the

TD-OCT images. Our TD-OCT helps investigate in

the senescence of leaves without destroying the

structure of the plant.

5 CONCLUSIONS AND FUTURE

PLANS

We studied the microstructural changes of the healthy

and unhealthy part of the bamboo leaves using the

OCT images. Environmental factors such as sunlight,

nutrients, and oxygen deficiencies affect the

morphological structures and the ability of the leaves

to produce chlorophyll. A significant change in the

extinction coefficient of the unhealthy part as

compared to the healthy part. This means that the

penetration of the light is possible due to the change

in the color. This is verified by measuring the RGB

data from the same region of interest for both parts. It

can be observed that the intensity and frequency

distribution of red and green data are similar from the

healthy leaves; while a significant shift in the red

color is observed in the unhealthy part.

To understand the color changes, the chlorophyll-

a content is measured. A 60% decreased was

observed in the chlorophyll-a content from the

unhealthy to the healthy part of the bamboo leaves.

This supports the microstructural changes observed

by the OCT images. Overall, an inverse correlation

was observed between the extinction coefficient and

the chlorophyll-a content of the bamboo leaves. With

the on-going development of the portable TD-OCT

scanner, we plan to extend the understanding

microstructural changes associated with the

senescence of leaves using our system.

ACKNOWLEDGEMENTS

J. Cadondon acknowledges support through the

DOST ASTHRDP and Enrichment Program. This

research was funded by the Commission on Higher

Education (CHED) of the Philippine Government for

the project entitled “Development of a Portable

Optical Coherence Tomography System for the

Evaluation of Human Skin Analogues”.

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118

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