Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

The overall goal of this procedure is to utilize synchrotron based x-ray micro tomography technology to explore the structure and function of vascular transport in plants. This is accomplished by first preparing samples for installation in a chuck holder or the live plant holder, ensuring that the portion to be scanned is as vertical as possible and taking necessary preliminary physiological measurements. The second step is to place the prepared samples or live plants into the A LS Beamline 8.3 0.2 hutch, and secure the hutch for scanning.Next.

After proper positioning of the sample, the scan is initiated. The final step is to utilize the workstation computers to normalize, reconstruct, and evaluate the quality of the scan before passing the data into a VISO for the 3D visualization process. Ultimately, x-ray micro tomography is used to reveal fine details of the interconnections and functional status of the water conducting vasculature in plants.

The main advantage of this technique over existing methods like serial sectioning and light microscopy, is that plant tissue can be explored in any orientation with unprecedented resolution. This method can help us understand key questions in the field of plant biology, from fundamental aspects of water transport in plants to drought, and freezing tolerance to how pathogens move systemically in host plants. This protocol as described, is designed for work at the advanced light source.

8.3 0.2. Beamline adaptations may be required for work at other synchrotron facilities. Be sure to follow the safety and radiation training required for use of these facilities to begin sample preparation for live plants.

First grow plants in approximately 10 centimeter diameter pots, ensuring that the main stem or other portion of the plant to be scanned is as centered as possible and oriented vertically in the pot. The physical dimensions of the HRCT instrument Hutch limits live plants to about one meter in height. As a consequence, imaging of live plants is best performed on seedlings or saplings.

Grown in small pots use a custom made rigid aluminum pot holder to mount the live potted plants. The top plate height should be adjusted to accommodate a range of pot heights. Here, the top of the plate is designed to align with the top of the soil surface and the plant protrudes from the center of the two-part plate.

Once mounted in the holder, measure the stem water potential using a SHO lander style pressure chamber or a clip-on leaf parameter to determine the physiological status of the plant prior to scanning. Twist a small piece of copper wire around the stem to serve as a fiduciary to ensure consistent scanning location on plants that will be scanned repeatedly. Now, place a thin walled acrylic cylinder over the plant on top of the aluminum plant holder and secure it in place with screws to stabilize the sample.

Additional plastic wrap paper towels and tape should be used to further minimize vibration and movement of plant parts, which can cause image distortions. Attach the custom pot holder to the air bearing stage and lock it into place positioned between the x-ray source and the imaging sensor and camera equipment. Be sure to position the stem as vertically as possible and center the sample on the magnetic chuck base to ensure it stays in the field of view During rotation, fresh plant material, typically stems or pets can be scanned after immediate removal from a live plant.

If the intent of the experiment is to visualize the entirety of the xylem network, water within the vessels must first be evacuated and replaced with air. To do this, mount the sample in a slander style pressure chamber and push compressed air or nitrogen through the sample at low pressure for approximately five minutes. Species will differ in the time required to evacuate the vessel network.

If the intent is to evaluate the extent of embolism formation in the fresh plant tissue, excise samples from the plant using a fresh razor blade making the cuts underwater. Next, wrap the sample in a layer of paraform to prevent desiccation during the scan mount the sample in a drill chuck fixed to a metal plate screwed into the air bearing stage center, and orient the sample vertically as previously described in order to ensure the sample remains in the field of view. To prepare samples from dried woody tissue begin by cutting samples to approximately six centimeters in length.

Select samples that are as straight as possible in the targeted scan region and have a diameter of about one centimeter. The next step is to slowly dehydrate the entire sample to ensure optimal tissue sample visualization and image contrast. Place the woody tissue sample into a drying oven at low temperature to slowly dry the sample without causing any cracking or splitting of the tissue.

In some situations, it may be desirable to have a fiduciary marker attached to the sample. This assures that subsequent dissection and visualization utilizing scanning electron microscopy can be oriented to specific points in the HRCT image. To do this affix a metal or glass bead or wire to the outside of the stem using parfum.

Finally, mount the sample in the drill check and center as described above prior to scanning. Determine the magnification that will work best for your application. The A LS Beamline 8.3 0.2 used here has the capability to scan with lenses with magnifications of two x five x and 10 x.

Set the x-ray energy to 15 kilo electron volts. Exposure times are generally dependent on the thickness and density of the sample and range from 100 to 1000 milliseconds. Choose an angular increment that is appropriate for your application.

Samples are rotated 180 degrees during a scan, and the number of images taken during the rotation can have a significant impact on the size of the dataset, length of the scan interval and final image quality. Typical scans are performed at 0.25 degree increments, yielding 513 images per scan. Shorter scan intervals can be achieved using the continuous tomography setting during which the sample continuously rotates while the images are captured for each scan, Brightfield and darkfield images must also be collected.

Brightfield images are images without the sample in the beam. These are often collected before and after the scan of the sample by horizontally translating the sample. Dark fields are collected by closing the x-ray shutter.

This measures the amount of signal the camera shows with no x-rays. Once the scan is complete, transfer the raw 2D TIFF images from the acquisition computer to a file server and then export to a computer to be used for data processing. Next, the images must be converted to a percent transmission scale.

The Beamline 8.3 0.2 has a custom background normalization plugin that can be downloaded and used with the freely available software packages. Image J or Fiji load the normalized images into the octopus software package, then reconstruct a 3D data set from the 2D raw TIFF image files using the designated processing steps. Next, the stack of images can be visualized in one of a variety of software packages.

Here the aviso software package is used, load datasets into system memory and display the sample in virtual transverse, longitudinal or radial slice orientations Because of the 3D attributes of the dataset, virtual slices through the sample can be rotated in any plane to align with the regions of interest. Once segmentation has been accomplished, it is possible to quantify the target plant structures or functional changes in volume, length, width, presence, or absence of water, air, et cetera. Synchrotron HRCT scans have been successfully implemented on a wide variety of plant tissues and species using Beamline 8.3 0.2, and have provided new insights into the structure and function of plant xylem at unprecedented resolution in 3D.

The visualization and exploration capabilities provided by the 3D reconstructions as seen here allow for precise determination of location and orientation of structures with the Xylem networks on both excise samples and in living plants. Here we see a 3D reconstruction of redwood stem subjected to drought stress and exhibiting both air and water-filled traches Once mastered, this technique can be performed in minutes if done properly Following this procedure. Other methods like scanning electron microscopy can be used to validate the structures we see inside the plants and come up with size thresholds, which are then fed into the processing programs that we use for the data analysis.