The goal of this study was to determine a more direct time saving method to visualize stems of cereals for cellular wall structure comparisons using 3D micro-CT imaging.
Micro-CT imaging of plants can be challenging due to the low level of x-ray absorption of the tissues (12). Our initial micro-CT (XT H 225, Nikon Metrology Inc., Brighton, MI, USA) scanning parameters were 75 kV tube voltage, 75 µA tube current, 708 ms exposure time, 720 projections and 4 frames per projection. The total scanning time was approximately 35 min for each specimen. The resulting images were grey, blurry, and undefined with no cell boundary details and limited contrast (Fig. 2: B & C). Only one peak was formed (Fig. 2A). Lowering the x-ray energy and exposure time for each sample improved the resolution in the low-density herbaceous plant tissue. Adjusting scanning parameters to optimal settings of 100 kV, 40 uA and exposure time of 354 with a magnification of 33.05 gave us a voxel size of 4.05 microns and resulted in a second peak in the grayscale (Fig. 2D). This second peak indicated a successful separation of the sample from the background (Fig. 2D) which would allow the tissues to be isolated using a suitable grey-value threshold (Fig. 2: A & D). The number of projections used was 360, with 16 frames per projection. These settings provided a good signal-to-noise ratio. No filter was used for the X-ray beam because it did not improve the quality of the scans.
To further widen the grey scale and to improve visibility of details, we introduced a commonly used contrast agent (Hexibrix 12.5% (v/v) Guerbet LLC, Bloomington, IN, USA) through a fresh stem using capillary action. Stem segments (3-5 cm) were partially embedded into the foam and filled with 400 ml of Hexibrix. The foam helped stabilize the stem for scanning. However, the contrast agent did not penetrate the stem section; contrast only coated and clumped on the outside surface. In addition, the physical stem length needed to be reduced for higher resolution. Optimized machine parameters included bringing the sample closer to the target resulting in a magnification of 33.05 and a voxel size of 4.05 µm. A straight stem segment with a diameter of 0.5-1 cm and length between 3-5 cm increased the likelihood of the 3D reconstruction software to identify the center of rotation.
After machine optimization, the samples remained blurry, possibly due to auto digestion and interior movement through liquid evaporation (13). Cellular degradation on a micro level during the duration of the scan is reasonable (i.e. evaporation), therefore, it was necessary to fix the tissues to preserve the morphological and molecular features of the tissue. We tested fixatives to assist in dehydrating the stems prior to imaging. In addition, the ability for a contrast agent to penetrate the cellular structures is dependent on proper fixation of the plant tissues (10, 14). Fixative selection is important, as there can be an interaction due to crosslinking between the fixatives and contrast agents (15). This interaction can cause the clumping of contrast in the tissues. The optimal combination of fixing and contrast agent to use are ones that have similar pH values (16). Not one individual fixative was recommended or preferred over another in the literature; therefore, we compared the images of samples prepared using four acidic fixatives. After the stems were cut to the appropriate size, they were immediately placed into 1.5-mL micro-centrifuge tubes, filled with one of the following four fixing agents: 70% ethanol (ETOH), formaldehyde alcohol acetic acid (FAA), Farmer, and Carnoy’s (Supplementary Table 1). All samples used fresh fixatives and no vacuum was applied during fixation. The samples were then stored at 4°C in fixative solution overnight and scanned the following day.
The addition of contrast improved the x-ray attenuation. Capillary action in a desiccator did absorb some of the contrast but it was insufficient to penetrate all the stem tissue. Different tissue reactions materialized according to the combination of fixative and contrast agent and the density of the tissues; one-day soak was sufficient for absorption of contrast after fixation to increase the x-ray attenuation into the internode whereas a two-day soak in contrast was needed for the thicker node material (Fig. 3 & 4). This difference is due to changes in the cross-linking of the fixative-contrast combinations within the organic tissue. These compounds bind to a variety of chemical groups in the tissues, often affecting the charge at the site of attachment (15). The internodal tissues fixed with FAA and Carnoys with contrast (Osmium Tetroxide or Phosphotungstate) were comparable (Fig. 3 & 4). When two combinations give comparable results, it is important to use the least toxic combination. The combination of FAA and phosphotunstate resulted in better images for the node sections while Carnoy’s and phosphotunstate worked better for the internode sections. FAA was chosen for its ability to absorb into the high-density cells (nodes) allowing the contrast to penetrate and distribute evenly. Even though FAA and Carnoy’s were comparable for the internodes, Carnoy's seemed to have better distribution of the contrast within the internodal tissues. There was no difference in x-ray attenuation between the two contrast agents; therefore, we chose to use phosphotunstate over osmium tetroxide because it was less toxic.
To further ensure the removal of all water out of the cells, the tissue was chemically dried with hexamethyldisilane (HMDS) (Fig. 3). Plant stems required a longer dry time then previously recorded (16). This phenomena may be because the structure of plant cell walls impedes water loss. Therefore, allowing the chemical drying agent to fully evaporate from the plant under the hood was required. The introduction of contrasting agents and chemical drying increased the signal to noise ratio of the grey values to above 100 with the peak just under 300 in both the node and internode (Fig. 5E & 6E). This process allowed the plant tissue to stop the auto digestion, absorb the contrast agent uniformly and dry the tissue. Internal structures such as the vascular tissue could now be differentiated.
The optimized micro-CT images were compared with environmental SEM (TM-3000, Hitachi, High-Technologies Corporation, Tokyo, Japan) images of paired stem sections. Similar levels of detail can be seen (Fig. 5 & 6) between the micro-CT and SEM 2D images. Both methods could differentiate between cell primordia within the parenchyma tissues. The successful separation of the cellular boundaries within the micro-CT images permits the study of the spatial organization of the tissues and allows for detailed 3D micro-CT images to be created. The resulting 3D images were used to develop a sophisticated 3D numerical model to predict structural behaviors (2).
Analysis of plant tissue
We believe this is the first time cereal nodal structures have been 3D imaged in detail. Niklas (17) discussed the physics for nodes in detail, but only described them as septa or diaphragm like. The introduction of transverse septa or nodes to the hollow tube increases the strength and flexibility of the tube-shaped stem (Fig. 1). These struts reduce the tube length, thereby, reducing regionally increasing stiffness of the tube. Nodes, solid amorphous tissue connecting long stem segments, can increase stiffness as much as 16 – 20%, even though they only contribute about 2% of the total stem weight (Niklas 1989). The internode, long segments of the stem, has the characteristic distribution of the strengthened tissues like vascular bundles equally distributed as far from the stem axis as possible with a hollow pneumatic center (18).
The micro-CT images allowed for clear visualization of cellular boundaries. The mechanical, parenchyma and lumen areas have distinct densities that are visualized by the micro-CT (Fig. 7). We did not find a statistical difference between the overall oat and wheat stem diameter (p=0.18). However, the thickness of the mechanical layer changed in size between species. This result has been seen between various wheat genotypes (19). Wheat clearly had larger vascular bundles composed of sclerenchyma in the mechanical layer, which could contribute to increased internodal strength compared to oat (20). Grasses, like cereals, use cylindrical geometry to maximize their strength and flexibility with the least amount of cellular tissue. A cylinder and to a greater extent, a hollow tube, is the most efficient geometric form to resist bending, elastic buckling, and torsion (21). Therefore, it is not surprising that plants use variations of this geometry in their growth.
In both cereals, two distinct rings separate from each other during scan preparation (Fig. 7D & H). The outer ring is not continuous, unlike the inner ring; an area can be clearly seen where the outer ring begins wrapping around the inner ring. This phenomenon is not noted in studies that used fresh material (22–25). The ring alignment overlap can be seen in the 2D cross sections of the micro-CT scans (Fig. 7D & H). This overlap could be an artifact from the direction in which the cells form the apical meristem align during development. A spiral pattern can be seen clearly during growth at the seedling stages, where oat at leaf arrangement is clockwise while wheat’s is counterclockwise.
Lateral and 3D images show various specialize structures with-in the nodal tissue that vary between species. First, oat nodes were found to be dense throughout the entirety of the nodal structure (Fig. 7A-C) while wheat contained large air pockets surrounding a center pith (Fig. 7E). These air pockets can also be seen on the lateral view (Fig. 7F & G). Secondly, the inner and outer thickness of nodal diaphragm or septa differed between oat and wheat (p= 0.031). The thinnest section of the nodal diaphragms is significantly greater in oat (1.94 ± 0.1 mm) (Fig. 7B & C) than wheat (1.58 ± 0.12mm) (Fig. 6F & G). The outer thickness of the node is also greater in the oat (2.94 ± 0.2 mm) than wheat (2.35 ± 0.3 mm). The nodes act as spring-like joints (26) where the spring constants of the nodes are directly related to the natural frequencies of vibration of the stems when set in motion (21, 26, 27). The differences in the thickness of the nodal diaphragms results in different spring constants. Wheat contains a lighter “spring” that is flexible, while the oat “spring” is thick and stiff. Wheat also contains equally spaced air spaces resembling flat discs that compress without damaging cellular tissue to add more flexing capabilities within the node (Fig. 7E-G). These multiple pneumatic air spaces and mechanical structures add additional strength (18).
Within a flexed grass stem, total strain energy is divided between the nodal diaphragms and the internodal walls where both structural elements operate as a single mechanical system (28). The internodal walls provide rigidity and strength, while the nodes contribute flexibility. The cellular makeup and distribution pattern of those cells is different for each structure.