2.1.

Optical Coherence Tomography

A Fourier domain OCT (FD-OCT) system¹² was set up in this work to achieve high resolution OCT imaging. The schematic diagram of our FD-OCT system is shown in Fig. 1 . The light source used in the system is an Integral OCT femtosecond laser (Femtolasers Produktions GmbH, Vienna, Austria) with a center wavelength of $820\mathrm{nm}$ and an optical bandwidth of about $120\mathrm{nm}$ . Identical microscope objective lenses $(\mathrm{NA}=0.3)$ were placed in the sample and reference arms to mitigate dispersion mismatch in the two arms. The laser light is coupled into a $2\times 2$ fiber coupler, which splits it equally into the reference and sample arms. Light reflected from the reference and sample arms is combined in the interferometer and measured in the frequency domain using a spectrometer (HR4000, Ocean Optics, Dunedin, Florida). The original optical spectrum is modulated at a frequency directly proportional to the optical path length between the reference mirror and the sample. The spatial reflectance profile of the tissue is obtained from an inverse Fourier transform of the optical spectrum.

Fig. 1

Experimental setup of the FD-OCT system used in this study.

The axial resolution of the OCT setup was measured to be $4\mu \mathrm{m}$ in free space. The lateral resolution was $9\mu \mathrm{m}$ , as determined with a United States Air Force target card. To minimize dispersion effects caused by additional optics (which will worsen the axial resolution), the beam in the sample arm was not expanded after the collimation lens to fill the back aperture of the objective lens. As a result, the measured lateral resolution was poorer than the theoretical resolution of $1.5\mu \mathrm{m}$ . A signal-to-noise ratio of $93\mathrm{dB}$ was achieved with this setup. The scan rate was about $1\mathrm{A}\text{-}\text{line}∕\mathrm{s}$ and was limited by the acquisition time of the spectrometer and the signal processing time for data scaling and Fourier transforms. The power delivered to the leaf sample was about $5\mathrm{mW}$ , which, together with the objective lens, gave an irradiance that was lower than the damage threshold of plant leaves.¹³ Furthermore, the laser pulse width was broadened significantly by dispersion after passing through various optical components. Consequently, the peak power was also not sufficient to damage the leaf samples.

2.2.

Plant Materials

Leaf samples from Oncidium orchid plants were used in this study. Leaf samples with differing symptoms were collected from both healthy and CymMV-infected plants. The main source of infection in the CymMV-infected plants is through the horticultural tools used in dividing plants and harvesting flowers. The plants were infected with CymMV for up to $6\text{months}$ . These leaf samples were first imaged using our FD-OCT system immediately after they were harvested to obtain cross sectional OCT images. After OCT imaging, part of the leaf samples was subjected to standard histological preparation (fixation, sectioning, and haematoxylin-eosin staining). Some of the leaf samples underwent cryohistology to examine their optical properties without staining. The sections were then examined under a bright-field or phase-contrast microscope. The remaining leaf samples were sent for ELISA tests to positively identify virus-infected samples. We note that ELISA tests are sensitive enough to detect the presence of virus due to the high-titer nature of the CymMV in Oncidium orchids.^{14, 15, 16}

3.1.

Optical Coherence Tomography Analysis

We first compare the OCT and bright-field histological images of a leaf sample from a healthy plant in Fig. 2 . The OCT tomogram in Fig. 2a is a raw false-color image depicting the interferometrically detected optical intensity backscattered from different depths in a leaf sample. This backscattered intensity is associated with the differences in the refractive index between the cellular components and its surrounding medium in the plant tissue. The axial depth from the surface of the leaf sample is corrected using the average refractive index of plant tissues.¹⁷

Fig. 2

Comparison between the (a) OCT image and (b) histological section of an orchid leaf sample. Scale bar corresponds to $100\mu \mathrm{m}$ . ue—upper epidermis, vb—vascular bundle, st—stoma, and mc—mesophyll cell.

Comparing the histological section of the leaf sample in Fig. 2b, the upper epidermis, vascular bundle, and mesophyll cells are clearly evident in the OCT image. The distinctive horizontally elongated shape of epidermal cells and the more rounded shape of mesophyll cells are readily visible in the OCT image. The nuclei of the plant cells are barely discernible due to the resolution limits of the system. It can also be seen that the OCT signal is reduced with increasing depth into the leaf sample. An imaging optical depth of approximately $400\mu \mathrm{m}$ was achieved with the OCT system. As the leaf contains mostly water, an average refractive index of 1.33 was assumed, which results in a physical penetration depth of approximately $300\mu \mathrm{m}$ in the leaf. The limited measurement depth is attributed to the attenuation of the incident light via scattering in the plant tissue¹⁸ and the sensitivity of the spectrometer.

We next compare the OCT images of leaf samples from both healthy and virus-infected plants in Fig. 3 . Three CymMV-infected leaf samples with differing symptoms were imaged with the OCT system. One of the virus-infected leaf samples has visible chlorosis, while the remaining samples have no visible symptoms, with one of them being a small young leaf. ELISA tests performed on these samples confirmed that they are infected with CymMV. The ELISA tests yielded positive CymMV infection with results of approximately $104\text{-}\mu \mathrm{g}∕\mathrm{g}$ fresh weight for all three leaf samples.

Fig. 3

OCT images of (a) a healthy young leaf, (b) a virus-infected leaf with chlorosis, (c) a virus-infected leaf without visible symptoms, and (d) a small young leaf with virus infection but no visible symptoms. Inset shows the magnified view of the upper epidermal layers of the leaf samples. Scale bar corresponds to $100\mu \mathrm{m}$ .

The OCT images in Fig. 3 clearly revealed the epidermal layer and the underlying mesophyll cells in all the leaf samples. It is noted that the mesophyll cells in two of the virus-infected samples [Figs. 3b and 3c] are larger compared to those of the healthy sample. This can be explained by the larger size of the two virus-infected leaf samples in Figs. 3b and 3c compared to the healthy leaf sample. The region beneath the epidermal layer of the young leaf sample in Fig. 3d appears to be highly scattering, although this is not seen in the OCT images of other virus-infected young leaf samples. The origin of the highly scattering region throughout the young leaf sample is presently unclear, but it could be due to structural damage in the mesophyll cells as a result of the virus infection.

A clear difference between the OCT images of healthy and virus-infected leaf samples becomes apparent when we examine the epidermal layer. The epidermal layers of virus-infected samples are found to be highly scattering compared to that of a healthy sample. In particular, the horizontally elongated structure of the epidermal cells is no longer visible in the virus-infected leaf samples. Optical sections from the bottom surface of the leaf samples were also obtained with the OCT system. To verify the results, we imaged at least ten other healthy leaves (both young and matured) and virus-infected leaves that are visually symptom-free. The OCT images of all the leaf samples consistently showed a highly scattering epidermal layer in virus-infected leaf samples and a clear epidermal layer in healthy leaf samples.

As a quantitative measure, the average intensity value of the epidermis (excluding cell walls) in the OCT images was used to compare between the healthy and virus-infected leaves. This value is proportional to the intensity of the incident light and the reflectivity of the tissue components in the epidermis. The average intensity value over five healthy samples is $0.16\pm 0.09$ (arbitrary units). By contrast, this value is $0.38\pm 0.22$ for the five virus-infected samples. The average intensity values in the epidermis of healthy leaves are close to the measurement noise floor of 0.15, indicating that there is little or no scattering in the volume of healthy epidermal cells. The virus-infected samples have significantly larger average intensity values due to increased scattering in the epidermal cell volumes.

Abnormal nutrition and environmental conditions such as nutritional imbalance, excess salts, high light intensity, and genetic disorders can result in virus-like symptoms on orchid plants. A stressed plant can easily be mistaken to be virus-infected with just visual inspection. To investigate if stressed plants without virus infection exhibit the same high scattering feature in their leaves as virus-infected plants, we imaged leaf samples of several stressed plants that suffered from a lack of water over a prolong period. Figure 4 shows the typical OCT image of leaf samples from stressed plants without virus infection. The optical sections of these leaf samples are found to be similar to those of healthy leaves in Fig. 3a. The stressed leaf samples did not have the highly scattering epidermal layer observed in the OCT images of virus-infected samples.

Fig. 4

Typical OCT image of leaf sample from virus-free but stressed plants. Scale bar corresponds to $100\mu \mathrm{m}$ .

3.2.

Histological Analysis

In this section, we investigate whether the highly scattering feature observed in the epidermal layer of virus-infected leaf samples is visible under bright-field histological observation. Histological sections of healthy and virus-infected leaf samples were examined. Figures 5a and 5b show the typical histological cross sections of healthy and virus-infected leaves, respectively. Morphological features such as the epidermal layers and vascular bundles are observed to be similar in appearance between the healthy and virus-infected samples. Although the bright-field images showed considerable details of cellular structures, there appears to be no discernible difference between the histological images of healthy and virus-infected samples. In particular, the highly scattering epidermal layers seen in the OCT images of virus-infected samples are not observed in the corresponding histological sections. One possible explanation is that the content within the leaf cells is altered or removed by the histological preparation process. To examine the plant cells in their original form, cryosections of the leaf samples were also taken without any fixation or staining. These cryosections were then mounted on microscope slides and observed under both bright-field and phase-contrast microscopes. We were also unable to observe any epidermal differences between the cryosections of healthy and virus-infected leaf samples.

Fig. 5

Histological sections of (a) healthy and (b) virus-infected leaf samples. Scale bar corresponds to $100\mu \mathrm{m}$ .