Impact of Micro and Nano Zinc Oxide Particles on Lentil Seed’s Internal Activity Using Biospeckle Optical Coherence Tomography (bOCT)

Abstract

The increasing use of nanoparticles (NPs) in various industries has intensified research into plant–NP interactions. NP properties significantly impact their cellular uptake and plant effects, highlighting the need for advanced monitoring techniques to understand their influence on plant growth and seed germination. This study uses biospeckle optical coherence tomography (bOCT) to investigate the size-dependent effects of zinc oxide (ZnO) NPs and microparticles (MPs) on lentil seed internal activity, visualizing dynamic changes under ZnO particle stress. ZnO was selected for its agricultural relevance as a micronutrient. Lentil seeds were submerged in ZnO particle dispersions (<50 nm, <100 nm, 5 μm, 45 μm) at concentrations of 0 (control), 25, 50, 100, and 200 mg/L. OCT structural images were obtained at 12.5 frames per second using a swept-source OCT (central wavelength 1.3 μm, bandwidth 125 nm, sweep frequency 20 kHz). OCT scans were performed before immersion (0 h) and 5, 10, and 20 h after lentil seed exposure to particle dispersion. The biospeckle image, representing dynamic speckle patterns characteristic of biological tissues, was calculated as the ratio of standard deviation to mean of 100 OCT structural images over 8 s. Biospeckle contrast was compared 0, 5, 10, and 20 h post-exposure. ZnO NPs <50 nm and 100 nm negatively impacted lentil seed biospeckle contrast at all concentrations. In contrast, 45 µm ZnO MPs significantly increased it even at 100 mg/L, while 5 μm MPs decreased biospeckle contrast at higher concentrations. bOCT results were compared with conventional morphological (germination percentage, growth, biomass) and biochemical (superoxide dismutase, catalase, and hydrogen peroxide) measurements. Conventional methods require one week, whereas bOCT detects significant changes in only five hours. The results from bOCT were consistent with conventional measurements. Unlike standard OCT, which monitors only structural images, bOCT is capable of monitoring internal structural changes, allowing rapid, non-invasive assessment of nanomaterial effects on plants.

1. Introduction

Nanoparticles (NPs) are particles with all three dimensions between 1 and 100 nm. They are made up of various materials, including pure metals such as zinc, gold, silver, iron, and titanium, their respective oxides and hydroxides, as well as non-metallic substances such as silica, polymers (e.g., polystyrene, poly (lactic-co-glycolic acid) or PLGA), and carbon-based materials [1]. Due to their minute size, NPs exhibit completely different characteristics from their larger counterparts, such as a significantly higher surface area-to-volume ratio, enhanced reactivity, distinct optical and electronic properties, extremely high stability, high adsorption capacity, and exceptional catalytic capabilities [2].

NPs originate from natural and engineered sources, and each contributes to their environmental presence. The natural sources of NPs include volcanic eruptions, forest fires, dust storms, and various biological processes, such as the breakdown of organic matter [3]. These particles have been integral to Earth’s ecosystems for millennia. In contrast, engineered nanomaterials have rapidly gained prominence due to their expanding industrial and technological applications in recent decades. The amount of engineered nanomaterials produced worldwide has been rising exponentially. From 2024 to 2030, the nanomaterials market is expected to expand at a compound annual growth rate (CAGR) of 15.0% from its estimated USD 12.42 billion in 2023 [4]. Concerns over the possible effects of these materials on the environment have been raised by their rapid development in use and manufacture. Zinc oxide (ZnO) NPs are among the most produced nanomaterials due to their exceptional sensing qualities and antibacterial traits, and extensive use in electronic sensors, optoelectronic devices, medical supplies, cosmetics, food packaging materials, and the agricultural sector [5]. Owing to the widespread use of products containing ZnO NPs, there is an estimated yearly global production of 550–33,400 tons, second only to TiO₂ NPs in terms of environmental presence [6]. For example, ZnO NPs were found in rainfall with 1.0 × 10⁵ particles per mL, and in natural rivers in Canada with 4.4 × 10⁵ particles per mL by Frechette-Viens et al. [7]. The concentration of ZnO NPs in wastewater entering a treatment plant in Maryland, USA, ranged from 20.0 ± 12.0 to 212.0 ± 53.0 μg/L, according to a similar study by Choi et al. [8].

Soil is the primary sink for both natural and engineered NPs, accumulating them through various processes such as material breakdown, atmospheric deposition, and agricultural applications [9]. NPs can interact with soil components and be absorbed by plant roots. As NPs can move through plant tissues, they may affect plant physiology, growth, and development [10]. Yang et al. reported the phytotoxic effects of ZnO NPs on Arabidopsis seedlings after an exposure period of 10 days at 40 mg/L and 60 mg/L concentrations [11]. Similarly, Keshta et al. reported higher toxicity of ZnO NPs on wheat (Triticum aestivum L.) germination and growth parameters after 3 days in comparison to bulk zinc with EC₅₀ value of 20.7 mg/L, indicating nano-specific toxicity [12]. However, Benavides-Mendoza et al. reported no significant negative impacts of ZnO NPs on lettuce after 64 days of transplanting at the 10⁻⁶ molar concentration range. The toxicity of these NPs to plants varies based on their physical and chemical properties, exposure conditions, environmental factors, and the specific species involved [13]. To evaluate phytotoxicity, these studies use a variety of techniques, such as morphological evaluations (seed germination, biomass, root, and shoot length), biochemical analyses (antioxidant enzyme activity, reactive oxygen species levels), and microscopy methods (transmission electron microscopy and scanning electron microscopy) to visualize the uptake of NPs. However, these approaches have drawbacks: the biochemical analyses need a destructive approach, the microscopy techniques do not offer real-time observations of NPs interactions with living plant tissues, and the morphological assessments may fail to detect subtle physiological changes and require an extended period. Despite extensive research, no existing method provides insights into the impact of NPs on plants at the critical pre-germination stage, revealing a significant methodological gap in understanding early plant–NP interactions. This complexity demands a careful evaluation of NP’s ecological risks across different scenarios and a real-time and fast monitoring technique [14,15].

Optical coherence tomography (OCT) is an interferometric imaging technique that offers ultra-high-resolution cross-sectional views with microstructure of biological tissue. Because of its non-contact and non-destructive nature, OCT is widely used in the medical sciences, including ophthalmology. Meanwhile, plant science research is also exploring its potential. OCT has been utilized for various purposes in plant science, such as non-invasive screening for disease in plant seed specimens, assessing germination rates of plant seeds, and detecting leaf spot diseases [16].

In recent years, dynamic optical coherence tomography (dOCT) techniques have advanced significantly, enabling visualization and quantification of motion and temporal changes within biological samples. These methods analyze temporal fluctuations of optical signals from intracellular organelle movements, offering insights into cellular mechanisms. For example, Thouvenin et al. developed a dynamic full-field OCT approach to reveal subcellular metabolic contrast in fresh ex vivo tissues by analyzing interferometric signal time dependence [17]. Additionally, dOCT has been applied to study plant-pathogen interactions, providing new perspectives on cellular and subcellular dynamics [18]. Biospeckle optical coherence tomography (bOCT), developed in our lab is part of the broader field of dOCT techniques. It utilizes speckle patterns and can monitor the dynamic response of plants beyond just cross-sectional images of the conventional OCT, offering real-time, non-invasive, and highly sensitivity advantages Compared to traditional methods, bOCT offers significant advantages in early detection of effects on seeds, providing real-time, non-invasive measurements of subtle physiological changes that may occur before visible germination. Our group has employed bOCT to visualize the effects of acid mine drainage (AMD) on rice and soybean seed germination within a much shorter time, 48 h and 72 h [19]. Additionally, we have used bOCT to investigate the impact of heavy metals such as zinc within 6 h [20].

The current study focuses on lentil (Lens culinaris), a very nutritious crop that is considered one of the most significant food crops in Asia because of its high protein and flavonoid content. As a mainstay of many Asian diets, lentils are crucial to the food security and agricultural economies of these Asian countries [21]. Lentils provide an excellent model to research the interactions between NPs and seed biology because of their quick germination cycle, well-defined seed coat structure, and flexibility to a wide range of environmental conditions [22].

We propose the application of bOCT to investigate the impact of micro and nano ZnO particles on lentil seed’s internal activity. This study aims to elucidate the potential effects and mechanisms of NPs interactions with seeds at the earliest stages of development, addressing a critical gap in our understanding of plant–NP interactions. Furthermore, we compare the results obtained from bOCT with the conventional measurements, seedling growth, biomass, and physiological parameters such as enzyme activities of superoxide dismutase (SOD) and catalase (CAT), as well as hydrogen peroxide (H₂O₂) levels conducted after 7 days of exposure.

2. Materials and Method

2.1. Nanoparticles

In the experiment, lentil seeds were exposed to different concentrations of 0, 25, 50, 100, and 200 mg/L ZnO NPs and microparticles (MPs), a range commonly used in various studies [23,24]. ZnO particles of sizes <50 nm (>10.8 m²/g surface area, >97% purity), <100 nm (10–25 m²/g surface area), and <5 µm (99.9% purity) were purchased from Merck Sigma–Aldrich, Japan, in dry powder form. Additionally, ZnO 45 µm particles in dry powder form were purchased from Goodfellow Japan with 99.9% purity. The emulsion solution containing 0.05% Tween-80, a non-ionic, biocompatible surfactant frequently used for dispersing hydrophobic particles in aqueous solutions, was made to overcome the particle’s hydrophobic features and achieve uniform dispersion. First, the solutions were stirred using a magnetic stirrer for 30 min and then sonicated at 28 °C for 20 min. The same protocol was repeated until all the particles were uniformly dispersed and no clusters of particles appeared in the solutions.

2.2. Plant Material

Lentil seeds (Lens culinaris) were purchased from a certified organic seed provider Greenfield Project Co., Ltd., Tokyo, Japan, and stored in a cool, dry area until use. To achieve the best germination, healthy seeds weighing about 30 mg each were chosen and surface sterilized. The sterilization process involved immersing the seeds in 2.5% H₂O₂ solution for 10 min, followed by three thorough rinses with distilled water. This standard procedure eliminates surface microorganisms, ensuring a clean experimental environment for accurate ZnO particle effect observations [25]. The seeds were then exposed to ZnO particle dispersion at 25, 50, 100, and 200 mg/L concentrations, with distilled water serving as the control. For the bOCT experiments, six lentil seeds were placed in each Petri dish, exposed to a 6 mL dispersion of each concentration of NPs, and maintained in a controlled growth chamber (MLR-351H, SANYO Electric Co., Ltd., Moriguchi City, Osaka, Japan). The chamber conditions were set to an air temperature of 25 °C/20 °C (day/night), light intensity of 260–370 μmol m⁻² s⁻¹/0 μmol m⁻² s⁻¹ (day/night), and relative humidity of 50–65%, following a 12 h/12 h day/night cycle. bOCT contrast images were captured at 0, 5, 10, and 20 h after exposure to ZnO particles to monitor the seed response at the early stages of NPs and MPs treatment.

2.3. Biospeckle Optical Coherence Tomography (bOCT)

2.3.1. Optical Experimental System

A schematic of the bOCT experimental system and its practical design, set up on an optical bench using optical fibers for image acquisition, is depicted in Figure 1. This system is a swept-source OCT system based on a Mach–Zehnder interferometer, utilizing a swept-source laser (HSL-2100-ST, Santec, Komaki City, Aichi, Japan) with a central wavelength of 1.3 µm and a bandwidth of 125 nm, total average power of 23.4 mW, and a sweep frequency of 20 kHz. The axial resolution of the system is 6 µm, and the lateral resolution is 39 µm. To illuminate the reference and sample arms independently, a 50/50, 1 × 2 optical coupler (TW1300R5A1, Thorlabs, Newton, NJ, USA) divides the light from the source into two beams. The reflected light in each arm was recombined using 1 × 2 optical couplers (TW1300R5A2, Thorlabs) after passing through the circulators (CIR-1310-50-APC, Thorlabs). A 50/50, 1 × 2 optical coupler was used to split the interference signal yet again to eliminate the source fluctuations. A balanced photodetector (PDB470C, Thorlabs) was then utilized for detecting the signal. A stepper motor stage (KXL06300-C2-F6A, Suruga Seiki, Shizuoka, Japan) was used to attach the reference mirror to modify the optical path length and acquire the interference signal. An OCT Probe unit (Santec, Aichi, Japan) was utilized to focus light onto the sample after light from the fiber was collimated by a fiber collimator on the sample arm.

To generate 3D OCT structural images, the light beam was scanned laterally in the x-y directions using a scanning probe consisting of galvano scanning mirrors. The data were acquired by custom-made LabVIEW software (LabVIEW ver.2012 National Instruments, Austin, TX, USA) followed by image analysis with MATLAB (MathWorks version R2016b, Natick, MA, USA) software. When scanning the probing beam with the galvano mirrors, only one lateral axis (x) was scanned to obtain a 2D image; a total of 100 frames, each with 3048 (z) × 1000 (x) pixels, were acquired at a B-scan acquisition rate of 12.5 frames per second over 8 s, where interframe variance frequency is 12.8 fps. These cross-sectional images represent the conventional OCT images, providing only the structural information. To determine the depth profile of the sample, the spectral interference signal obtained as a function of wavelength from the swept source was converted to frequency, wave vector, or k-space and then Fourier-transformed.

We utilized 1.3 µm near-infrared light, which is known for its ability to penetrate deeper into biological tissues while minimizing localized absorption, to evaluate its effects on living cells. The power incident on the seed surface was approximately 100 µW, corresponding to a radiant dose of 1.39 mJ/cm² for the 8 s exposure. This dose is considerably below the levels typically associated with laser-induced effects in plant tissues. Given these parameters, we assumed that no living cells in the seeds would be adversely affected by the exposure. While there are no universally established safety limits for seed irradiation, studies on various plant species support this assumption. For instance, Metwally et al. reported positive effects on seed germination and seedling growth using laser treatments with much higher power levels, up to 10 mW, which is 100 times greater than the power used in our study [26].

2.3.2. Biospeckle and Contrast

When coherent light, such as laser light, illuminates a plant, the optically rough structures within the plant scatter the light, resulting in a granular speckle pattern known as biospeckle. This biospeckle pattern is formed due to the random interference of the scattered light [27]. For a static object, the intensity of the speckle pattern remains constant over time. However, in dynamic objects, where the scattering centers are always moving, as in the case of seed organelles, the scattering pattern changes during cytoplasmic flow, cell division, water transport, and cell expansion, resulting in intensity fluctuations. These dynamic speckle patterns, referred to as ’biospeckle,’ can be used to characterize the activity of the biological sample and are evident in the bOCT structural images, where intensity variations can be observed at each pixel along the temporal axis due to the internal activities of the cells. The degree of these fluctuations is quantified by biospeckle contrast, γ, which is defined as the ratio of the standard deviation of the intensity to the mean value at each pixel along the temporal axis over the entire scan. This biospeckle contrast is calculated using the following equation:

$$\gamma \left(x,z\right)={\displaystyle \frac{1}{<I_{OCT}\left(x,z\right)>}}{\left[{\displaystyle \frac{1}{N}} {\sum }_{j=1}^N{\left\{I_{OCT}\left(x,z, t_j\right)-<I_{OCT}\left(x,z\right)>\right\}}^2\right]}^{{\displaystyle \frac{1}{2}}},$$

(1)

where

$${<I}_{OCT}\left(x,z\right)> ={\displaystyle \frac{1}{N}} {\sum }_{j=1}^N{I}_{OCT}\left(x,z, t_j\right),$$

where x stands for the lateral coordinate, z for the depth coordinate, j is the scan number, t_j is the time corresponding to the jth scan, and N indicates the total number of scans.

Higher biospeckle contrast signifies larger temporal fluctuations, indicating a higher internal activity within the measured seed, and lower biospeckle contrast corresponds to a reduction in temporal fluctuations, indicating lower internal activity. Therefore, the biospeckle contrast can be employed as a measure to evaluate the response of the seeds to external environmental changes.

2.4. Growth Parameters Analysis

2.4.1. Germination Percentage and Seedling Growth

Conventional measurements were performed after 7 days of exposure to validate bOCT results. Lentil seeds were considered to be germinated when their radicle extension reached approximately 2 mm, generally occurring within 24 h [22]. Non-viable seeds were those that showed no signs of growth or infection by fungus. With the use of ImageJ software (version 1.54d, NIH, Bethesda, MD, USA), the lengths of the seedling’s roots and shoots were measured. Three replicates of ten seeds, each of the ZnO NPs (<50 nm) and MPs (45 µm) at 25 mg/L and 100 mg/L treatment concentrations, were used to initiate the seedlings. To evaluate the overall performance of the seeds under various treatments, the germination percentage (%) and vigor index were calculated, respectively, and defined by

$$Germination Percentage (\%)={\displaystyle \frac{\mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s}}{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s}}}\times 100.$$

(2)

$$\mathrm{V}\mathrm{i}\mathrm{g}\mathrm{o}\mathrm{r} \mathrm{I}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}=\left[\mathrm{A}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{R}\mathrm{o}\mathrm{o}\mathrm{t} \mathrm{l}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}+\mathrm{A}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{S}\mathrm{h}\mathrm{o}\mathrm{o}\mathrm{t} \mathrm{L}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h} \left(\mathrm{c}\mathrm{m}\right)\right]\times \mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\%$$

(3)

2.4.2. Biomass Measurement

Root and shoot biomass measurements provide crucial insights into the plant water content and growth responses to the various treatments. In this study, ten lentil seeds were germinated for each treatment of ZnO NPs and MPs of size <50 nm and 45 µm at concentrations of 0 (control), 25, and 100 mg/L, with three replicates per condition. After germination, the seedlings were washed to remove contaminants or residual chemicals and gently pat-dried. The shoots and roots were separated, and their fresh weights were measured using an analytical balance (AUX 320, UniBloc, Shimadzu Corporation, Kyoto City, Japan). To determine dry weights, the samples were oven-dried (SOFW-450S, AS ONE, Osaka City, Japan) at 105 °C, initially for 2 h, then at 80 °C until they reached a constant weight for 72 h.

2.5. Oxidative Stress Response Analysis

To compare the bOCT results with the conventional measurements, an assessment of the antioxidative enzyme activity in lentil seedlings exposed to ZnO NPs (<50 nm) and MPs (45 µm) after 7 days of germination was conducted. Lentil seedlings were exposed to ZnO NPs and MPs at concentrations of 0 (control), 25, and 100 mg/L for 7 days. Following exposure, the seedlings were thoroughly rinsed with distilled water and weighed. The samples were then homogenized in phosphate buffer (0.01 mol/L, pH 7.4) using a pre-chilled mortar and pestle in an ice bath. The homogenate was centrifuged at 3000 rpm at 4 °C for 15 min, and the resulting supernatants were used for subsequent analyses. A spectrophotometer (UVmini-1240, Shimadzu Corporation, Kyoto, Japan) was employed to measure the absorbance values for various assays. The hydrogen peroxide (H₂O₂) content was determined using a commercial assay kit (CL-204, National Diagnostics, Atlanta, GA, USA), with the absorbance measured at 560 nm. Superoxide dismutase (SOD) activity was assessed using a commercial kit (S311-WST, DOJINDO, Kumamoto, Japan), with the absorbance measured at 450 nm. Both assays were performed according to the manufacturer’s instructions. Catalase (CAT) activity was measured spectrophotometrically, following the standard procedure described by Aebi (1984) [28]. The decrease in absorbance was monitored at 240 nm over 3 min, with measurements at 1 min intervals. The decline in absorbance corresponds to the decomposition of H₂O₂ by CAT.

2.6. Statistical Analysis

For each treatment condition involving four types of ZnO particles at concentrations of 25, 50, 100, and 200 mg/L, six seeds were used as samples. The data were collected from 100 optical coherence tomography (OCT) scans per seed at four time points: 0, 5, 10, and 20 h of exposure. Initially, all OCT image data were obtained using custom-made software developed in LabVIEW (LabView ver.2012 National Instruments, Austin, TX, USA). The bOCT data analysis was performed using MATLAB (R2016b) software from acquired OCT data, which provides complex data processing and interpretation. All graphs and histograms for data visualization were made and modified using Origin 9.5, which produced understandable visual depictions of the outcomes. Statistical analysis was performed using EXCEL (Microsoft 365, version 2412, Redmond, WA, USA) to determine the significance of the variations between the treatment and control groups. Each triplicate sample’s results are shown as the mean ± standard deviation of the mean, with Student’s t-test (p < 0.05) used to identify significant differences.

3. Results

3.1. bOCT Measurements

3.1.1. Comparison of Biospeckle Contrast

Internal structural changes in lentil seeds during germination were investigated in the current study using optical coherence tomography (OCT). OCT is increasingly being used in environmental sensing and agriculture despite its traditional employment in the in vivo viewing of internal biological structures. Figure 2a–l shows the conventional OCT cross-sectional images and bOCT contrast images exposed to ZnO MPs (45 µm) and NPs (<50 nm) at 25 mg/L concentration with the vertical axis representing the depth and the horizontal axis representing the lateral position. Here, in Figure 2a–d with bright areas denoting strong reflectivity and dark regions denoting weak reflectivity, the OCT structural images show the interior laminar structure of the seed. The high reflectivity of the seed coat and epidermis allow them to be visible clearly at the top of the structural image.

The OCT structural images of seeds exposed to ZnO MPs (45 µm) for 0, 5, 10, and 20 h do not show significant variations. Beyond the laminar architecture, tiny granular features called speckles can also be seen in the OCT images. The smaller internal scattering microstructures in the seed, such as mitochondria, golgi bodies, and maybe chloroplasts, are the origin of these speckles. The motion of various constituents of the cell, possibly induced by mechanisms such as cell growth, division, and cytoplasmic flow, influence the biospeckles observed in bOCT contrast images.

bOCT contrast images were calculated according to Equation (1) from the series of OCT frames captured during each treatment before and after ZnO MPs (45 µm) and NPs (<50 nm) at 25 mg/L concentration exposure for 0, 5, 10, and 20 h, as illustrated in Figure 2e–l. As seen by the color bar, in these bOCT contrast images, red indicates a larger biospeckle contrast, and blue shows smaller biospeckle contrast. Blue-dominated areas indicate less internal activity in the seed, while intense red regions in bOCT images indicate higher internal activity.

Here, in Figure 2e–h, bOCT images of lentil seed exposed to ZnO MPs with a 45 µm size at 25 mg/L showed a notable and significant increase in biospeckle contrast at 5, 10, and 20 h of exposure time, as the red color density increased significantly. However, as the blue color density is more evident in bOCT images, Figure 2i–l showed a significant reduction in the biospeckle contrast of lentil seed as a result of ZnO NPs, with a particle size of <50 nm being exposed for 5, 10, and 20 h.

Figure 3a,b present a comparison of bOCT contrast images for various ZnO particle sizes at 25 and 100 mg/L concentrations and exposure times. The images are arranged in a grid, with rows representing exposure times (0, 5, 10, and 20 h) and columns showing different particle sizes (control, 45 µm, <5 µm, 100 nm, and <50 nm). The control group uses only distilled water without particles, while the other columns show ZnO particles of different sizes. Figure 3a shows results for a 25 mg/L concentration, while Figure 3b presents results for a 100 mg/L concentration. In Figure 3a, columns 1, 2, and 3, corresponding to the control, 45 µm, and <5 µm, respectively, show a more pronounced increase in red color density. This indicates higher intensity fluctuations compared to those in columns 4 and 5, which correspond to particle sizes 100 nm and <50 nm. This observation suggests that larger particles (45 µm and <5 µm) and the control group exhibited more significant changes in bOCT contrast over time compared to the smaller NPs (100 nm and <50 nm) at the 25 mg/L concentration.

In Figure 3b, which represents the results for the concentration of 100 mg/L, a similar tendency is observed when comparing those for the NPs and MPs. Across all columns, the 45 µm MPs (column 2) show the highest red color density in the exposed lentil seed’s bOCT contrast images, indicating the most pronounced intensity fluctuations. Columns 4 and 5, representing the smaller NPs (100 nm and <50 nm), display more blue color density at this concentration. These observations collectively suggest that particle size plays a critical role in determining bOCT contrast, with larger MPs, particularly the 45 µm size, producing the most significant temporal changes in biospeckle contrast. It is important to note that only bOCT contrast images show this minute change in all activities. On the other hand, conventional OCT structural images failed to reveal these minute internal changes, highlighting the enhanced sensitivity of the bOCT technique in capturing dynamic structural changes within the seed.

3.1.2. Quantitative Analysis

A comprehensive quantitative analysis of bOCT images was performed to examine variations in the internal activity of the seed. This procedure involved calculating the normalized contrast and average local contrast values. Specific regions of interest (ROIs) between the shallow and deeper regions of the seed were determined for the purpose of average local contrast (Figure 4). Six ROIs were selected from each bOCT image. A grand average of average local contrast was then determined from six ROIs. The average local contrast of each treatment at various periods was normalized against the values obtained at 0 h (pre-imbibition) to account for relative variations in individual seeds and the average normalized contrast for each treatment were then calculated by averaging these normalized contrasts over a sample of six seeds. This two-step procedure substantially reduced the impact of seed-to-seed variability and provided a more robust analysis of internal seed activity variations.

Figure 5a–d demonstrate the obtained bespeckle contrast results with the horizontal axis representing the time in hours of exposure at which bOCT scans were taken and the vertical axis being averaged normalized contrast. The bOCT results, shown in Figure 5a, revealed that even at lower concentrations (25 mg/L), the averaged normalized contrasts of lentil seeds for <50 nm and <100 nm-sized ZnO NPs were significantly lower than those for 45 µm-sized particles only after 5 h of exposure. Additionally, at 5, 10, and 20 h of exposure, respectively, the averaged normalized contrast or internal activity of lentil seeds exposed to 100 mg/L 45 µm-sized ZnO particles increased by 7.6%, 11%, and 25.5% relative to those of control seeds shown in Figure 5c.

On the other hand, during the exposure period, particles <50 nm exhibited a significant decrease in normalized contrast of 14%, 17%, and 21%, while <100 nm particles showed a decrease of 12%, 15%, and 16%, respectively, in comparison to those of the control. Similarly, during the same exposure time, lower concentrations (25 mg/L) of ZnO NPs (<50 nm) showed decrements of 7%, 4%, and 5%, and <100 nm particles showed a 15%, 15.5%, and 19% decrease in the normalized contrast of lentil seeds.

Additionally, ZnO particles sized at 5 µm did not exhibit any significant effects at concentrations up to 50 mg/L. However, at higher concentrations of 100 mg/L and 200 mg/L, as shown in Figure 5c,d, there were significant reductions in the normalized contrasts, indicating an adverse effect. Specifically, at 100 mg/L, the reductions were 12%, 12%, and 9% at 5, 10, and 20 h, respectively. At 200 mg/L, the reductions increased to 21%, 22%, and 29% for the same exposure times. The percentage changes in averaged normalized contrasts for lentil seeds, exposed to ZnO particles of sizes <50 nm, 100 nm, 5 µm, and 45 µm at concentrations of 25, 50, 100, and 200 mg/L at 5, 10, and 20 h of exposure, are described in

Table 1. Change in average normalized contrast relative to control condition of lentil seeds.

Particle Size	Concentration (mg/L)	Average Normalized Contrast (Percentage Change) ± Error
		5 h (%)	10 h (%)	20 h (%)
<50 nm	25	−7 ± 0.73	−4 ± 0.32	−5 ± 0.35
	50	−17 ± 2.21	−13.8 ± 1.73	−8.7 ± 0.82
	100	−14 ± 1.81	−17 ± 2.23	−21 ± 2.71
	200	−14 ± 1.77	−16 ± 2.02	−16.6 ± 2.08
100 nm	25	−15 ± 3.17	−15.5 ± 3.24	−19 ± 3.89
	50	−17.4 ± 3.26	−20 ± 0.00	−23 ± 0.01
	100	−12 ± 0.15	−15 ± 0.00	−16 ± 2.04
	200	−24.2 ± 3.03	27.2 ± 3.39	−31.73 ± 3.96
<5 µm	25	−5 ± 0.53	−5 ± 0.56	−2 ± 0.26
	50	0.9 ± 0.23	3 ± 0.35	−2 ± 0.42
	100	−12 ± 1.15	−12 ± 1.53	−9 ± 0.85
	200	−21 ± 2.73	−22 ± 2.80	−29 ± 3.61
45 µm	25	4 ± 0.09	0.7 ± 0.00	3 ± 0.01
	50	1.4 ± 0.13	2.8 ± 0.24	6.5 ± 0.31
	100	7.6 ± 0.09	11 ± 0.00	25.5 ± 0.01
	200	1.8 ± 0.02	−0.05 ± 0.00	−0.9 ± 0.01

.

Interestingly, 100 nm-sized NPs consistently showed a significant negative impact from lower concentrations (25 mg/L) to 200 mg/L, with a reduction of up to 31.7% at 20 h of exposure at 200 mg/L. The results of the bOCT revealed that ZnO MPs (45 µm) have a significant positive effect on the internal activity of lentil seeds up to 100 mg/L. Furthermore, at 200 mg/L, no adverse effects were observed. In contrast, <5 µm-sized ZnO MPs showed statistically significant (p < 0.05) negative impacts at 100 and 200 mg/L concentrations, while lower concentrations did not show any significant impacts. On the other hand, ZnO NPs (<50 nm) had an adverse effect, even at 50 mg/L. The detailed analysis of the size-dependent effects and their potential mechanisms is explored in Section 4.

3.2. Comparison with Conventional Measurements

Following exposure to ZnO NPs and MPs, a number of conventional morphological and physiological measures were carried out to confirm and interpret the results from the bOCT examinations. The germination rate, shoot length, root length, fresh weight, dry weight, and enzyme activity of the seedlings were among the conventional measurements. To compare the bOCT results with these conventional measurements, we selected two types of particles: nano-sized particles (50 nm) and micro-sized particles (45 μm). The comparison was conducted at two distinct concentrations: 25 mg/L and 100 mg/L. For this focused analysis, we selected these specific particle sizes and concentrations from the four concentrations and four-particle sizes used in the overall bOCT experiment.

3.2.1. Morphological Parameters

In this study, the conventional morphological measurements, such as germination percentage, seed vigor index, root and shoot length, and biomass measurements, were performed after 7 days of exposure to ZnO NPs <50 nm and MPs 45 μm at 25 mg/L and 100 mg/L concentrations. It is important to note that while root and shoot length measurements took 7 days, bOCT provided short-term measurements within just 20 h. Figure 6a,b show the root and shoot length measurements at 100 mg/L and 25 mg/L concentrations, respectively, for both particles <50 nm and 45 μm, with the horizontal axis representing the particle size and the vertical axis representing the length of root and shoot in centimeters. As shown in Figure 6a, the root and shoot length results are consistent with bOCT measurements. At 100 mg/L, ZnO NPs-exposed lentil seeds showed a significant decrease (p < 0.05) in root and shoot length, while ZnO MPs-exposed lentil seeds demonstrated an increase. In contrast, at 25 mg/L concentration, no significant difference was observed for both NPs and MPs, as shown in Figure 6b.

Figure 6c shows the germination percentage and seed vigor index. A significant reduction in germination percentage and vigor index was observed in 100 mg/L ZnO NP-exposed seeds, indicating the adverse effect of ZnO NPs. This finding is well supported by the lower normalized contrast of bOCT shown in Figure 5c, which was obtained within only 20 h. In contrast, 45 µm-sized ZnO particles showed a significant increase in the germination rate and root and shoot length, acting as a beneficial micronutrient source without inducing the toxicity or stress associated with NPs, probably due to their slower dissolution and reduced cellular penetration.

Figure 7a–d show the biomass measurements of root and shoot fresh weight and dry weight exposed to MPs and NPs at 25 and 100 mg/L concentrations with the horizontal axis representing the particle size and the vertical axis weights in gram. The fresh weight (FW) and dry weight (DW) of seedlings serve as critical parameters for evaluating the immediate effects of nanomaterials on plant growth. These measurements are widely recognized as essential indicators in assessing the acute impact of nanomaterial exposure on seedling development patterns. Depending on factors such as particle size, concentration, and chemical composition, FW and DW can reveal both positive and negative influences of nanomaterials [29]. The effect of ZnO NPs and MPs at 25 mg/L and 100 mg/L on root and shoot fresh weight of lentil seedlings is shown in Figure 7a,c. A significant reduction in root fresh weight at 25 mg/L and shoot fresh weight at 100 mg/L were observed for NPs exposed seedlings. In contrast, a significant increase in root fresh weight at 100 mg/L for micro ZnO particles was observed, indicating a positive impact of MPs which is consistent with our bOCT results obtained within a short time. These could be attributed to NPs-induced stress, impaired water uptake, and disrupted cellular processes due to the particle’s small size and high reactivity. No significant difference was observed in the dry weight of seedlings, as shown in Figure 7b,d. However, the dry weight of seedlings exposed to NPs was observed to be less compared to those treated with MPs at both 25 and 100 mg/L concentrations.

3.2.2. Physiological Parameters

To compare the obtained bOCT results with physiological changes within the plant, a series of antioxidative enzyme assays and oxidative damage assessments were performed for ZnO particles sized <50 nm and 45 μm at concentrations of 25 and 100 mg/L, as shown in Figure 8a–c).

Figure 8a represents the Catalase enzyme activity (CAT) for lentil seeds exposed to ZnO NPs (<50 nm) and MPs (45 μm), with the horizontal axis showing concentrations in mg/L and the vertical axis showing CAT content (U/g-min). At 100 mg/L concentration, CAT activity increased significantly (p < 0.005) for ZnO NPs. In contrast, 45 μm ZnO MPs at 100 mg/L showed a different trend, as the CAT activity decreased significantly (p < 0.05) compared to the control. At 25 mg/L, no significant changes were observed for CAT activity compared to that of the control.

Similarly, Figure 8b shows the Superoxide dismutase (SOD) activity test results. SOD activity is measured as percentage inhibition, denoted on the vertical axis, while concentrations are presented on the horizontal axis. The percentage inhibition for SOD activity represents the percentage reduction in the rate of superoxide anion-mediated reduction in a chromogen compared to that of the control. It indicates the enzyme’s effectiveness in the dismutation of superoxide anions. The SOD activity results were similar to the CAT activity results. At the higher concentration of 100 mg/L, a significant increase in SOD percentage inhibition was observed in lentil seeds exposed to smaller <50 nm-sized particles. For the lower concentration, no significant changes were observed in SOD activity percentage inhibition.

In Figure 8c, the hydrogen peroxide (H₂O₂) content (μmol/g-FW) is shown on the vertical axis and ZnO particle concentrations are shown on the horizontal axis. An increase in H₂O₂ was observed; 11.8% and 13.6% for 45 μm ZnO particles at 25 and 100 mg/L, respectively, while for nano <50 nm particles, increases of 17% and 25.4% in H₂O₂ were observed at 25 and 100 mg/L concentrations, respectively. This suggests that the H₂O₂ content increased substantially, leading to enhanced CAT and SOD activity (measured as percentage inhibition). The high concentration of ZnO NPs induced oxidative stress, triggering an antioxidant response dominated by CAT and SOD to neutralize the excess H₂O₂. The contrasting responses between NPs and MPs highlight the size-dependent effects of ZnO particles on plant antioxidant systems, with NPs generally inducing a more severe oxidative stress response, particularly at higher concentrations.

4. Discussion

The primary aim of this study was to examine the differential effects of ZnO NPs and MPs on lentil seed germination and early seedling development using a novel optical method, namely biospeckle optical coherence tomography (bOCT). In this study, bOCT provided rapid and reliable measurements, offering significant insights into NP-induced changes in internal seed activity, which were compared to conventional physiological parameters such as germination rate, root length, shoot length, biomass accumulation, and antioxidative enzyme activities. Furthermore, the technique is capable of monitoring the dynamic changes within the seed during germination could be adequately investigated by bOCT contrast within a short period in vivo, while the conventional OCT monitors just structural images.

In addition, conventional measurement studies typically observe responses over 7 days, using a common concentration range from 1 mg/L to 200 mg/L [30]. When calculated over the entire 7-day period, these concentrations translate to total exposure doses of 168 mg/L-h to 33,600 mg/L-h. This range represents the broader spectrum of NP dosages commonly used to evaluate impacts on plants. In our study, we observed significant responses within only 20 h, allowing us to calculate total exposure doses as 500 mg/L-hours to 4000 mg/L-h for the respective concentrations. Despite the shorter observation period, our total exposure doses fall within a comparable range to the broader range observed in conventional studies. This highlights the sensitivity of our method in detecting early biological responses to ZnO NPs and MPs.

The bOCT results presented in Figure 5a–d reveal a complex, size-dependent impact of ZnO particles on the lentil seed internal activity. Notably, nano-sized particles (<50 nm and 100 nm) demonstrated a strong negative impact, while micro-sized particles showed varying effects. The 100 nm ZnO particles consistently exhibited the most significant negative impact on the seed internal activity across all concentrations. This pronounced effect may be attributed to an optimal size for cellular uptake and interaction with biological systems. At this size, particles may efficiently penetrate seed coats and cell membranes, leading to increased intracellular accumulation and potential toxicity. The high surface area to volume ratio of 100 nm particles likely enhances their reactivity, further contributing to their negative impact [31]. Interestingly, <50 nm particles, while also showing negative effects, appeared less detrimental than 100 nm particles. This difference could be due to quantum effects potentially altering particle behavior and interactions. Włodarczyk et al. also reported the more beneficial effect of <50 nm particles in comparison to 100 nm ZnO NPs [32]. Additionally, smaller particles may have a greater tendency to aggregate, possibly reducing their effective surface area and cellular uptake [33]. This complex behavior aligns with studies showing non-monotonic dependence of nanocrystalline ZnO properties, such as elastic modulus, on crystallite size. The non-linear relationship between particle size and properties suggests that the effects of ZnO nanoparticles on seeds may not follow a simple trend as particle size changes, further emphasizing the intricate nature of size-dependent effects at the nanoscale [34,35].

The 5 μm ZnO particles demonstrated intermediate effects, showing negative impacts only at higher concentrations. This size is comparable to the average pore size of lentil seed coats (4–8 μm) [36], suggesting that these particles may partially block seed pores without fully penetrating the seed structure. This blockage could impede nutrient and water uptake and lead to the observed negative effects at higher concentrations. In contrast, 45 μm ZnO particles exhibited a significant positive effect on seed internal activity up to 100 mg/L, with no adverse effects observed even at 200 mg/L. The larger size of these particles likely prevents them from penetrating the seed coat or cellular structures. Instead, they may act as a stable, external source of zinc, a known micronutrient for plants. These findings are consistent with studies that highlight zinc as a critical micronutrient for plant development when applied in suitable forms and concentrations [37]. This could enhance plant metabolism, promote early growth processes, and improve photosynthetic performance, leading to larger biospeckle contrast.

Table 2. Effects of different particle sizes and concentrations on plant morphological parameters and physiological parameters.

Treatment	Concentration (mg/L)	ANC ± SD (20 h)	Length (cm) (7 Day)		FW (g) ± SD (7 Day)		DW (g) ± SD (7 Day)		Physiological Parameters (7 Day)
			RL ± SD	SL ± SD	SFW ± SD	RFW ± SD	SDW ± SD	RDW ± SD	SOD ± SD	CAT ± SD	H2O2 ± SD
Control	0	1.77 ± 0.02	4.70 ± 0.39	2.96 ± 0.40	0.29 ± 0.01	0.54 ± 0.03	0.052 ± 0.02	0.063 ± 0.03	38.39 ± 3.47	19.16 ± 1.66	0.59 ± 0.05
45 µm	25	1.83 ± 0.03	4.86 ± 0.49	2.99 ± 0.51	0.32 ± 0.03	0.58 ± 0.02	0.054 ± 0.02	0.072 ± 0.005	42.91 ± 15.23	21.53 ± 4.66	0.66 ± 0.12
	100	2.22 ± 0.02	5.39 ± 0.23	3.81 ± 0.22	0.31 ± 0.02	0.66 * ± 0.10	0.062 ± 0.02	0.071 ± 0.03	36.67 ± 5.07	10.33 * ± 2.51	0.67 ± 0.11
<50 nm	25	1.69 ± 0.03	4.50 ± 0.54	2.83 ± 0.39	0.30 ± 0.02	0.45 * ± 0.01	0.050 ± 0.01	0.055 ± 0.02	45.04 ± 2.53	27.69 ± 5.96	0.69 ± 0.11735
	100	1.39 ± 0.04	3.99 ± 0.34 *	2.04 ± 0.46 *	0.26 ± 0.07	0.59 ± 0.02	0.049 ± 0.01	0.069 ± 0.01	67.27 * ± 20.54	36.60 * ± 3.89	0.74 ± 0.10

Abbreviation: ANC = Averaged normalized contrast, SD = Standard deviation, RL = Root length, SL = Shoot length, SFW = Shoot fresh weight, RFW = Root fresh weight, SDW = Shoot dry weight, RDW = Root dry weight, SOD = Superoxidase dismutase, CAT = Catalase, H₂O₂ = Hydrogen peroxidase (* represent statistical significance of data p < 0.05).

demonstrates the correlation between bOCT results for 20 h exposure at 25 and 100 mg/L with 7-day conventional measurements.

For example, the lowest normalized contrast value (1.39 ± 0.04) and the highest value (2.22 ± 0.02) for <50 nm and 45 μm ZnO NPs, respectively, at 100 mg/L correspond to the most significant reductions and increment in length, fresh weight, and dry weight, as well as the levels of antioxidative enzymes. This strong correlation underscores the predictive potential of early bOCT measurements for long-term effects on plant growth and stress responses. Morphological experiments focusing on 45 μm and <50 nm, ZnO particles revealed a clear reduction in germination rate, root length, and shoot length for seeds exposed to ZnO NPs (<50 nm), particularly at higher concentrations (100 mg/L). This negative impact aligns with previous studies highlighting NP toxicity in plants often attributed to the smaller size and higher reactivity of NPs [29,38]. In our study, ZnO NPs measured approximately <50 nm, and 100 nm, while MPs were <5 μm and 45 μm. The size-dependent characteristics observed could be explained; as NPs can penetrate the seed, whereas larger MPs are unable to achieve such penetration. Furthermore, biomass measurements (fresh and dry weights) supported the ZnO NPs (<50 nm) induce stress, as evidenced by the significant reduction in seedling fresh weight. This stress could be due to impaired water uptake, cellular damage, or metabolic disruption caused by NP’s interaction with seed tissues and the accumulation of Zn²⁺ ions [39]. In contrast, MP (45 μm) treated seedlings showed enhanced biomass accumulation, reinforcing the idea that MPs have a more beneficial influence on plant growth. Importantly, our use of bOCT enabled us to detect these significant changes within only 20 h of exposure much earlier than the conventional growth parameter measurements that typically require a week.

The changes in antioxidative stress markers further explain the different effects observed between NPs (<50 nm) and MPs (45 μm). ZnO NPs led to a significant increase in H₂O₂ content, CAT activity, and SOD activity, particularly at 100 mg/L. The increase in H₂O₂ suggests that NPs exposure triggers oxidative stress through the generation of reactive oxygen species (ROS). This is a common stress response in plants exposed to metal-based NPs, as their small size allows them to interact directly with cellular components, disrupting normal cellular homeostasis and leading to ROS production. The accumulation of Zn²⁺ ions from the dissolution of NPs within the seed tissues may further contribute to this oxidative stress. The elevated CAT and SOD activity in NP-treated seedlings indicates an upregulation of the plant’s antioxidant defense system in response to this oxidative stress. CAT is responsible for breaking down H₂O₂ into water and oxygen, helping to mitigate the damage caused by excess ROS [11].

In contrast, the antioxidative response to ZnO MPs (45 μm) is significantly different. Seedlings exposed to MPs at 100 mg/L exhibited lower CAT activity, while SOD activity and H₂O₂ levels showed no statistically significant difference compared to that of the control. The lower CAT activity suggests that less H₂O₂ is being produced, indicating that oxidative stress is less severe in MP-treated seedlings. This might be explained as the larger MPs have limited cellular penetration and do not generate ROS to the same extent as NPs. As a result, the antioxidant system does not need to be as actively engaged, leading to lower CAT activity. The consistent SOD activity further suggests that the oxidative stress is minimal, and the plant is not experiencing significant disruption in its ROS detoxification pathways. The size-dependent effects of ZnO particles on antioxidative stress markers highlight the importance of particle size in determining the level of stress experienced by plants. Smaller NPs are more reactive and can penetrate cellular structures, leading to greater oxidative stress and an upregulation of the antioxidant defense system, particularly CAT. However, larger MPs seem to induce significantly less stress, reflected by the lower levels of CAT activity and stable SOD function [40].

The use of a 1.3 µm wavelength laser source in our bOCT system allows for precise visualization of the internal activity of lentil seeds exposed to ZnO NPs. Significant changes in internal activity were recorded at 0, 5, 10, and 20 h, with the control samples demonstrating a clear increase in internal activity over time. This consistent rise in internal activity highlights the sensitivity of the bOCT technique to biological processes within the seeds. Through a detailed analysis of the speckle patterns in both the treated and control samples, we were able to create an internal activity baseline that is not a result of motion artifacts from NPs but rather directly correlates with the physiological responses of the seeds. Similar findings have been reported in a study utilizing bOCT to investigate the effects of polyethylene microsphere (PEMP) exposure on lentil seedlings [41]. Additionally, research by Li et al. demonstrated the efficacy of bOCT in assessing the effects of AMD exposure on soybean and Kaiware daikon, reinforcing the applicability of our method in elucidating the effects of various stressors on seed physiology. These studies collectively support our findings, highlighting the robustness of the bOCT technique in monitoring the internal activity of seeds under the different environmental stressors [42]. Therefore, the observed alterations in biospeckle contrast during the exposure periods are indicative of the seed’s physiological responses to ZnO NPs exposure, confirming that the internal activity changes are a direct result of the seed’s inherent biological processes.

These findings highlight that bOCT is an effective technique for early detection of NP effects on seed physiology, providing insights before visible germination. The ability to detect changes at such an early stage shows the potential feature of this method in agricultural and environmental research. In addition, this non-invasive method allows real-time monitoring of internal seed activity, offering a unique perspective on environmental stressor’s impact on plant development.

5. Conclusions

The present research proposes the bOCT to monitor the effects of NPs and MPs on the internal activity of lentil seeds. The novel application of bOCT provided rapid, non-invasive insights into the early stages of seed-NP interactions, revealing significant changes within only 5 h of exposure. The current study revealed that ZnO NPs (<50 nm, <100 nm) induce more pronounced stress responses in lentil seeds compared to larger ZnO MPs, due to their ability to penetrate the seed coat through pores ranging from 4 to 8 μm in size and potentially interact with cellular structures, generating Zn²⁺ ions that cause damage. This size-dependent effect was evident in germination percentage, seedling growth parameters, and antioxidative enzyme activities. While NPs showed potential phytotoxic effects, MPs demonstrated a more beneficial influence, possibly serving as a slow-release source of zinc micronutrients. The proposed bOCT technique successfully observed the size-dependent impacts of NPs at an early stage, even before germination. Significant effects of NPs and MPs of ZnO were observed through the traditional measurements after 7 days of exposure, and the speed, reliability, and significance of the proposed approach were demonstrated. Furthermore, the technique is capable of monitoring the internal activities of seeds during germination, which could be adequately investigated by bOCT contrast within a short period in vivo, while conventional OCT monitors only structural images. Further studies are needed to explore the long-term effects of these interactions and to optimize the application of bOCT for broader agricultural practices.