Non-Destructive Direct Pericarp Thickness Measurement of Sorghum Kernels Using Extended-Focus Optical Coherence Microscopy

Abstract

Non-destructive measurements of internal morphological structures in plant materials such as seeds are of high interest in agricultural research. The estimation of pericarp thickness is important to understand the grain quality and storage stability of seeds and can play a crucial role in improving crop yield. In this study, we demonstrate the applicability of fiber-based Bessel beam Fourier domain (FD) optical coherence microscopy (OCM) with a nearly constant high lateral resolution maintained at over ~400 µm for direct non-invasive measurement of the pericarp thickness of two different sorghum genotypes. Whereas measurements based on axial profiles need additional knowledge of the pericarp refractive index, en-face views allow for direct distance measurements. We directly determine pericarp thickness from lateral sections with a 3 µm resolution by taking the width of the signal corresponding to the pericarp at the 1/e threshold. These measurements enable differentiation of the two genotypes with 100% accuracy. We find that trading image resolution for acquisition speed and view size reduces the classification accuracy. Average pericarp thicknesses of 74 µm (thick phenotype) and 43 µm (thin phenotype) are obtained from high-resolution lateral sections, and are in good agreement with previously reported measurements of the same genotypes. Extracting the morphological features of plant seeds using Bessel beam FD-OCM is expected to provide valuable information to the food processing industry and plant breeding programs.

Keywords: Bessel beam; Fourier domain optical coherence microscopy; higher-order-mode fiber; long-period grating; pericarp; phenotyping; sorghum; spatial resolution.

Figure 1

Schematic of the ultrahigh-resolution Fourier domain optical coherence microscopy setup. The inset shows the far-field beam profile of the LP₀₂ (Bessel) beam (scale bar 1 mm).

Figure 2

Images of the sorghum grains used in this study: BTx2928 (left) and RTx430 (right).

Figure 3

Widefield epifluorescence (a,d), two-photon microscopy (b,e), and high-resolution OCT (c,f) images of an RTx430 seed (a–c) and a BTx2928 seed (d–f). The widefield epifluorescence shows red fluorescence from the pericarp of the seed under a 40× objective. The two-photon images show red fluorescence in red, and green fluorescence, as well as second-harmonic radiation generated by the starchy endosperm in green. The red fluorescence in the widefield and multiphoton images allows for clear demarcation of the pericarp. The OCT images display the OCT signal, which originates mainly from the pericarp in a false color representation (FIJI “fire” color scheme). The scale bars in each image represent 50 µm.

Figure 4

Cross-sections from Bessel-beam FD-OCM tomograms of (a) BTx2928 and (b) RTx430 seeds. Scale bar: 0.5 mm. The “fire” color-scale from FIJI is used for the false color scheme in the monochrome images (pixel values are scaled according to the logarithm of the OCT signal), helping slightly to enhance small signal details. The position of the xy, xz, and yz cross-sections are indicated with dotted yellow lines in the each of the other sections.

Figure 5

Sketch of side and top cross-sections of a sorghum seed, indicating the pericarp, endosperm, embryo, hilum, and stylar regions. The latter two are on opposing sides of the seed, and we use the midline connecting these two regions to define the positions where we measure pericarp thickness, i.e., at ~30-degree intervals starting at ~30 degrees. The zero- and 180-degree positions are excluded from the measurement as around these positions, more irregularities are found.

Figure 6

Lateral (a) and axial (b) profiles from different positions in an RTx430 seed (red traces) and a BTx2928 seed (blue traces). (a) The pericarp thickness in the lateral profiles (OCT signal normalized to the peak value in the profile) is determined by the distance between where the signal rises through the 1/e threshold and falls below the 1/e threshold again. The black dotted lines are placed at 0 and 1/e, allowing them to indicate the thickness measurement. The upper traces are vertically offset by 1 from the lower traces. (b) The pericarp thickness in the axial traces is determined by the distance between the center of two Gaussian peaks (shown as the dotted traces) matched to the OCT peaks corresponding to the upper and lower boundary of the pericarp, or to the outer edges of the OCT signal peak when the two boundaries cannot be distinguished. Additionally, here, the normalized traces are each vertically offset by 1.

Figure 7

Box-and-whisker plots combining all pericarp thickness measurements with (a) 11 µm and (b) 3 µm lateral resolutions. The p-value obtained from Student’s t-test for the large-field-of-view measurements is 3.2 × 10⁻¹⁹ (a), and for the zoomed-in measurements (b) the p-value is 1.9 × 10⁻²⁹.

Figure 8

Plot showing the variation in pericarp thickness within each seed measured using the zoomed-in tomograms. Red points are for the RTx430 seeds, blue points are for the BTx2928 seeds, and points that are vertically aligned are for the same seed. Horizontally, the seeds are displayed according to the order in which they were measured. Open circles show the average for each individual seed. Boxes and whiskers show the median and quartiles for each seed, where 2 out of 200 measurements could be considered outliers. The red and blue dotted lines show the average pericarp thickness from the zoomed-in lateral measurements for the RTx430 and BTx2928 seeds, respectively. The black dashed line shows the threshold value that can be used to classify the seeds (see text for classification rules). For comparison, the open (red and blue) diamonds show the pericarp thickness values extracted from axial measurements.

Figure 9

Pericarp thickness compared to seed size (a) and position (b) along the seed perimeter. Red symbols are derived from RTx430 seed data and blue symbols from BTx2928 data.