Environmental Pollution Assessment with Indicator Plant Under

Ozone Gas Atmosphere by Using OCT

Hayate Goto

and Tatsuo Shiina

Chiba University, YayoiCho, Inage-ku, Chiba-shi, Chiba, Japan

Keywords: Environmental Assessment, Ozone, OCT, Field Measurement, Transparency, Leaves, Laser.

Abstract: Measuring plants’ sensitivity to environmental stresses can help us understand the environmental and

ecological conditions in the area. Optical Coherence Tomography (OCT) can visualize and evaluate the

internal plant structure quantitatively. In this study, as a preliminary step to assess the atmospheric

environment by field measurements of plants using OCT, the influence of the ozone gas and the effect of the

plant-clearing agent in OCT measurement were evaluated. The plant-clearing agent makes the internal

refractive index uniform and allows evaluation of the extinction coefficient from the leaf’s full cross-section

image. The results showed an increase in the extinction coefficient and its palisade thickness. The extinction

coefficient significantly changed between before and after exposure to ozone gas to 16.9 ± 6.2 [/mm] from

12.6 ± 3.42 [/mm]. This result indicates that OCT can measure plant responses to environmental changes

quantitatively. Field measurement of plants by OCT will allow environmental assessment anywhere in a short

time.

1 INTRODUCTION

In recent years, toxic substances in the air have

become high enough to affect plants and animals in

some urban areas (Masui et al., 2021, Graham et al.,

1998). One method of environmental assessment is to

use “indicator plants” that are sensitive to changes in

the atmospheric environment. This method does not

need expensive equipment. In addition, indicator

plants can be evaluated by visual process or by using

the conventional technique. If the atmosphere

becomes dry, the chlorophyll content in plants’ leaves

decreases. Spectroscopic observation can observe

those decreases (Kwartiningsih et al., 2021).

However, the reduction of chlorophyll can also be

caused by nutrient deficiencies and insect damage.

Spectroscopic observations can also be used to

observe plants’ responses that lead to pigment

changes, biochemical changes, and inhibition of

photosynthesis. Because of the simultaneous

occurrence of different plant stressors, it is not easy

to elucidate the causal stress.

If a plant experiences environmental stresses, the

effect of this stress is often found in the changes in

https://orcid.org/0000-0001-5387-9109

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

the plant’s internal structure. For this reason, close-

range remote sensing for observing plant growth is a

practical approach to evaluating environmental

stressors by measuring plant conditions. Optical

Coherence Tomography (OCT) is a remote sensing

technique that uses near-infrared light to visualize the

internal structures of living organisms. It has the

advantage over other methods, such as MRI, X-ray,

etc., in obtaining internal structures by being

inexpensive, compact, and can be used in the field

(Wijesinghe et al., 2017, Lee et al., 2019). This

research discusses the feasibility of in-situ

observation of the atmospheric environmental stress

on plants by measuring the change in leaves’ internal

structure.

One problem with OCT is that the measurement

depth is limited due to light scattering due to the non-

uniformity of the refractive index of a plant leaf’s

tissue (Wit et al., 2020). To match the internal

refractive index, we used a plant-clearing agent with

a refractive index close to the plant tissue that can

penetrate inside the plant (Villani et al., 2013). We

discussed the advantages of tissue clearing in field

observations with OCT.

Goto, H. and Shiina, T.

Environmental Pollution Assessment with Indicator Plant Under Ozone Gas Atmosphere by Using OCT.

DOI: 10.5220/0011679400003408

In Proceedings of the 11th International Conference on Photonics, Optics and Laser Technology (PHOTOPTICS 2023) , pages 34-39

ISBN: 978-989-758-632-3; ISSN: 2184-4364

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

We focused on ozone gas as environmental stress

on plants. Ozone gas is relatively high near industrial

and urban areas. Since high ozone concentrations can

affect plants, quantifying the effect of ozone gas on

plants is an essential aspect that this work wants to

establish. In this work, we exposed white clovers, the

ozone indicator plant, under a high concentration of

ozone gas and measured changes in the leaf’s internal

structure by using our original OCT. These results can

be used to infer ozone concentration in a

measurement area.

This work aims to evaluate the plant growing

environment by using OCT in orchards or polluted

areas. The OCT that was developed in our laboratory

can evaluate quantitatively by only point

measurement with the discussion of their optical

properties. The proposed method has the advantage of

easy environmental assessment anywhere.

2 METHOD

The OCT system used in this study is developed for

plant measurement in our laboratory, and its

configuration is shown in Fig.1(a). Fig.1(b) shows the

optical probe and its’ measurement scene. The size of

this OCT is shown in Fig.1(c), and it is small enough

to be easily taken it outside and runs on a DC battery.

This OCT, a TD-OCT, incorporates a rotation

mechanism in the reference optical path, and the

optical path length changes linearly with time. This

OCT is constructed to evaluate changes in the plant’s

internal structure by point measurement. Leaves are

quantitatively evaluated by acquiring A-scans in field

measurements. We select the wavelength of the light

source to be 1310 nm, which has a low absorbance for

chlorophyll and a local minimum absorbance for

water. The optical probe was designed to be small.

The probe is small enough to be positioned anywhere

near the leaf and at any angle during field

measurement. Table 1 shows the specification of the

OCT system. A-scan signals are acquired at 25 Hz,

and each A-scan is the average of 16 measurements

to reduce noises.

In the OCT measurement with a plant-clearing

agent, the leaves were placed between two acrylic

plates with a window in the measurement area (Fig.

1b). This method needs to measure the same position

of the leaf before and after the transparency process.

For this reason, the plant-clearing agent is filled into

the window, with the leaf in between two acrylic

plates. We used Visikol (Visiko Inc.) as the plant-

clearing agent, which has a refractive index

equivalent to plant tissue and makes the internal

refractive index uniform as it penetrates the interior

of the leaf. A uniform refractive index reduces

internal scattering and allows light to reach deeper

inside the plant tissue.

Figure 1: a) The configuration of the OCT system; b)

scanning of mounted transparent leaf (left) and probing of

leaves exposed to ozone (right); c) control box of the OCT

system.

Table 1: Specification.

Item specification

Center Wavelength

SLD Output

1310 nm

15 µW

FWHM 53 nm

Axial Resolution

Lateral Resolution

A-scan Rate

14.2 μm

10 µm

25 Hz

Average Times 16

Focal Length

5.12 mm

0.13

Beam Diameter 0.65 mm

OCT Size 198 × 168 × 98 mm

Probe Size φ6 mm×9 mm

SLD

Fiber coupler

Transparent leaves

Leaves exposed to ozone

Sample optical path

Reference optical path

Environmental Pollution Assessment with Indicator Plant Under Ozone Gas Atmosphere by Using OCT

(a): Before transparency process.

(b): After transparency process.

Figure 2: Transparency of Dracena leaves.

In the case of white clover, measurements are made

without cutting the leaves and keeping the plants in

pots so that the temporal changes of the leaf’s internal

structure due to the effect of ozone gas can be

measured with the same leaf. White clover was grown

in an incubator at 20°C, and the ozone concentration

in the incubator was approximately 0.21 ppm. In

addition, the blue and red light of the cold-cathode

lamp is turned on 15 hours in a day. Leaf

measurements were taken every 12 hours after

exposure to ozone gas.

3 RESULTS

3.1 Transparency Process of Dracaena

Leaves

Figures 2 (a) and (b) show the results of Dracaena

leaves before and after the transparency process with

Visikol, respectively. The x-axis in the figure

represents the horizontal point (B-scan direction), and

the y-axis is the depth direction (A-scan). OCT light

is illuminated from the top of the leaf’s surface. The

intensity of interference light is indicated by the color

bar. Furthermore, the intensity of interference light is

obtained by subtracting the background light, and

performing focal length correction, distance squared

correction, and logarithmic transformation from the

obtained original OCT signals.

The result before the transparency process is shown

in Fig.2(a). The adaxial surface is visualized and a

part of the abaxial surface is visualized, too. In the

Figure 3: Averaged intensity change with depth in Figure 2.

leaf’s interior, the signal from the layer near the

adaxial surface is partially visualized, while the layer

on the abaxial surface is hardly visualized. In the

result after the transparency process with Visikol

shown in Fig. 2(b), the entire adaxial surface is

visualized, but the abaxial surface is not. In the leaf’s

interior, the signal is obtained throughout the leaf’s

full cross-section image. The area indicated by the red

arrow in Fig. 2(b) has the signal of the leaf veins, and

the signal is stronger than in the other areas. The

signal of the abaxial surface is only obtained at the

right end part, and the signal disappears to the left of

3.0 mm of the x-axis. Similarly, the signal also

disappears at both ends of the adaxial surface. This

disappearance of the abaxial surface is caused by

delaminating the surface due to the transparency

process.

The adaxial surface position in Fig. 2 is aligned at

the same depth, and then the intensity changes with

depth direction are averaged in the horizontal

direction. In this case, both ends of the leaf and the

PHOTOPTICS 2023 - 11th International Conference on Photonics, Optics and Laser Technology

(a): Before exposure to the ozone gas.

(b): After exposure to the ozone gas.

Figure 4: White Clover under ozone gas.

veins are excluded from the averaging procedure.

Figure 3 shows these results. The y-axis shows the

average of the logarithmic intensity, and the x-axis

shows the depth. The blue line shows the results

before the transparency process, and the orange line

shows the results after the transparency process with

Visikol. Before the transparency process, the peak

intensities in the adaxial and abaxial surfaces can be

seen. After the transparency process, the abaxial

surface signal disappears. Inside peaks are obscure,

and its attenuation is monotonous.

The plant-clearing agent reduces the internal

refractive index differences. Since the plant-

clearing agent increases the light transmission of the

leaves’ interior, the veins’ location and thickness of

the layer become clear. In Fig.3, the transparency

process made the constant attenuation. Therefore,

the extinction coefficient can evaluate from the

leaf’s full cross-section signal. The use of the plnat-

clearing agent increases the OCT signal near the

abaxial axis side of the leaf. It can evaluate the

change of light transmission due to disease or

environmental stress in the field measurement.

3.2 Measurement of White Clover

Under Ozone Gas Atmosphere

Figure 4(a) shows the result before the white clover

was exposed to ozone gas, and Figure 4(b) shows

the result after 12 hours under ozone gas exposure.

In the same way as Fig.2, these figures show the

results of modified intensity by subtracting the

background light, applying focal length correction,

distance squared correction, and logarithmic

transformation from the obtained OCT signals. The

entire adaxial surface was visualized before and

after exposure to ozone gas, and the abaxial surface

was not visualized. The interface between the

epidermis layer and the palisade tissue became

clearer after exposure to ozone gas.

Figure 5: Intensity change with depth in Fig 4.

Figure 6: Extinction coefficient of multiple leaves in

palisade tissue.

Similar to Fig. 3, the surface in Fig. 4 is aligned

and the A-lines are averaged, except for the position

of the veins. Figure 5 shows these A-line average

results. The blue curve in the figure shows the result

024681012

Extinction Coefficient (/mm)

Leaf Number

Environmental Pollution Assessment with Indicator Plant Under Ozone Gas Atmosphere by Using OCT

before exposure to ozone gas, and the orange curve

shows the results after 12-hour exposure to ozone

gas. The interface of the palisade and spongy tissues

(at the 0.17 mm depth position in Fig.5) is almost

unchanged before and after exposure to ozone gas.

However, the attenuation of the signal in the

palisade tissue is increased by exposure to ozone

gas. The signal below the palisade tissue is

unchanged. The effect of ozone gas on plant leaves

appears in the palisade tissue (Thomson et al., 1966,

Hartikainen et al., 2020).

We measured 11 white clover leaves exposed to

ozone gas for 12 hours. Figure 6 shows extinction

coefficients in the palisade tissue. The extinction

coefficient is the slope of the black dotted line in Fig.

5. The x-axis in Fig. 6 indicates the leaf number, and

the y-axis shows the extinction coefficient. The blue

and orange circles in the figure represent the

measurement before and after 12 hours of exposure

to ozone gas. The two vertically aligned blue and

orange circles are the results of the same leaf.

The results show an increase in the extinction

coefficient of the palisade tissue in most leaves. The

mean value of the extinction coefficients before and

after exposure to ozone gas is 12.6 ± 3.42 [/mm] and

16.9 ± 6.2 [/mm], respectively. After exposure to

ozone gas, the extinction coefficient increased, and

the standard deviation is larger. A one-tailed t-test

was performed on this result as p = 0.003 (p<0.05).

These findings confirm that ozone destroys the

palisade tissue, and these changes can be evaluated

quantitatively from OCT signals.

4 CONCLUSIONS

In this study, we investigate the effect of the plnat-

clearing agent on the OCT images and the ozone gas

on the plant tissue to assess the environmental

contamination of ozone by OCT for the purpose of

future field measurements. The transparency

process increased and homogenized the internal

OCT signals from leaf measurements. The ozone

gas affects the epidermis tissue of white clover and

significantly increases the extinction coefficient

obtained by OCT in the palisade tissue.

Soaking the leaves in Visikol, the signal only

visible near the epidermis layer became uniformly

visible from the adaxial surface to the abaxial

surface. The position of the veins, which had

originally been unclear, could be confirmed, too. In

addition, the extinction coefficient inside the leaf

becomes uniform due to the decrease of the

refractive index difference by the penetration of the

plant-clearing agent. It makes it possible to evaluate

the change of extinction coefficient over the leaf’s

full cross-sectional signal. The transparency process

can confirm the disease or environmental stress,

which changes the transmittance of light. Since the

plant-clearing agent can make the leaf’s full cross-

sectional signal clear, diseases or environmental

stresses affecting the whole region of interest or

region of the leaf where we don’t know are affected

can be detected by the proposed method. Because

this method can detect by point measurement, OCT

can quantitatively and speedily assess

environmental conditions.

When plant leaves are exposed to high

concentrations of ozone gas, the palisade tissues are

destroyed. It has been difficult to clearly distinguish

these changes from mere OCT images. By

averaging each A-scan and comparing before and

after exposure to ozone gas, a clear difference in the

extinction coefficient in the palisade tissue appeared.

On the other hand, the spongy tissue was almost the

same before and after the ozone gas exposure. In

addition, we measured multiple leaves, which

confirmed that palisade’s extinction coefficients

predominantly changed before and after exposure to

the ozone gas by t-test.

In the leaf measurements with transparency

process and exposure to ozone, the changes can be

confirmed by A-scan, as shown in Figs. 3 and 5.

Thus, it is possible to confirm the effect of ozone

when white clover leaves from areas with high and

low concentrations of ozone are observed using

point measurements by this OCT method. Since this

result shows that our portable OCT can detect the

change in the leaf’s interior, OCT can estimate the

environmental conditions by measuring indicator

plants. In addition, the OCT with additional

functions can observe changes in the internal

structure of plants, and more accurate

environmental evaluation can be performed. For

example, polarized OCT, which can observe

separately p-polarization and s-polarization, can

capture changes in the internal polarization state of

plants, and b-OCT, which visualizes the speckle

variation with time, can evaluate plant activity

(Silva et al., 2021). If this method is established, this

portable OCT can be a useful instrument to assess

the identification or predict comprehensive

environmental stress.

PHOTOPTICS 2023 - 11th International Conference on Photonics, Optics and Laser Technology

ACKNOWLEDGEMENTS

This work was supported by JST, the establishment

of university fellowships towards the creation of

science technology innovation, Grant Number

JPMJFS2107.

REFERENCES

Masui, N. et al. (2021), Ozone disrupts the

communication between plants and insects in urban

and suburban areas: an updated insight on plant

volatiles, Journal of Forestry Research, 32, 1337-

1349

Graham, J.A. et al. (1998), Toxicology of ozone as

characterized by laboratory animals and extrapolated

to humans, Studies in Environmental Science, 72,

465-481

Kwartiningsih, E. et al. (2021), Chlorophyll Extraction

Methods Review and Chlorophyll Stability of Katuk

Leaves (Sauropus androgynous), Journal of Physics,

1858, 012015

Wijesinghe, R.E. et al. (2017), Optical coherence

tomography integrated, wearable (backpack-type),

compact diagnostic imaging modality for in situ leaf

quality assessment, Applied Optics, 56, 9, D108-

D1114

Lee, J. et.al (2019), On-Field In situ Inspection for

Marssonina Coronaria Infected Apple Blotch Based

on Non-Invasive Bio-Photonic Imaging Module,

IEEE Access, 7, 148684-148691 de Wit, J et.al (2020),

Quantification of plant morphology and leaf thickness

with optical coherence tomography, Applied Optics,

59, 33, 10304-10311

Villani, T.S. et.al (2013), An improved clearing and

mounting solution to replace chloral hydrate in

microscopic application, Applications in plant

sciences, 1(5), 1300016

Thomson, W.W. et.al (1966), EFFECTS OF OZONE ON

THE FINE STRUCTURE OF THE PALISADE

PARENCHYMA CELLS OF BEAN LEAVES,

Canadian Journal of Botany, 44, 12

Hartikainen, K. et.al (2020), Elevated temperature and

ozone modify structural characteristics of silver birch

(Betula pendula) leaves, Tree Physiology, 40, 4, 467-

483

Y.S.K De Silva et.al (2021), Positive and negative

phenotyping of increasing Zn concentrations by

Biospeckle Optical Coherence Tomography in

speedy monitoring on lentil (Lens culinaris) seed

germination and seedling growth, Plant Stress, 2,

100041.

Environmental Pollution Assessment with Indicator Plant Under Ozone Gas Atmosphere by Using OCT