Imaging Live Trees: Early Experiments

March 20, 2023

Imaging live trees at the resolution required to observe features at the cellular scale is challenging. There are many conventional and advanced measurements that are able to provide information at this cellular scale, but few can be used on living trees. Instead, samples must be first sectioned from the tree before being imaged.

Here is a selection of the techniques we are using in our research, and some examples of the images produced.

Light Microscopy and Electron Microscopy

Light optical microscopy and scanning electron microscopy are approaches used to image small structures on the surface of sections at high magnification. Light optical microscopy relies on lenses to focus light and make small regions visible to the eye (or a camera). Scanning electron microscopy uses a focused beam on electrons as its ‘light’ source, with electromagnetic lenses used to focus the beam, and a black and white image is formed based on the number electrons reflected off a sample.

Using both techniques we produce very similar kinds of images; the images below show sections of maple stem. Using these microscopy techniques, we can resolve the key features of the xylem, the region which occupies most of the stem cross-section. We see ray parenchyma, which run radially out towards the bark, the vessels, which run vertically, forming tubes through which sap is transported, and the fibres which fill most of the interstitial space and provide structural support.

Light optical (left) and scanning electron (right) micrographs of the stem of an Acer saccharum (sugar maple) sapling showing the cellular structures within the xylem. (Credit: Tenaya Driller).

We use these images to obtain basic structural information about the maple stem, including the sizes of the different cell types and their distributions, and at higher resolutions we can observe and characterise the small pits in cell walls that provide connections between neighbouring cells.

We have also investigated the use of different staining techniques to help identify cell types. Maple trees have two different types of fibres, which are indistinguishable by eye, but serve differing roles in the xylem and have different cell wall compositions. To distinguish between the types of fibre, staining(whereby light optical samples are pre-treated with different chemicals to highlight specific features) can be used to highlight fibres with more or less lignin (a protein) in their cell wall. The figure above shows such a staining, where fibres with less lignin appear blue, though the technique still needs some refinement.

3D X-ray Micro Tomography

X-ray micro-tomography (microCT) is a technique that uses X-rays to create cross-sections of a physical object. These images can be used to recreate a 3D image of the object. Bench-top microCT units can provide high resolution scans, though imaging time is long and their small apertures mean we must section branches from a tree in order to image them.

We are using x-ray micro-tomography to understand the distribution of sap and gas throughout the tree’s xylem (water conducting (vascular) tissue of plants). We focus on xylem embolisms (gas bubbles within xylem cells). MicroCT imaging provides good contrast between gas and solid/liquid material, and the position of embolisms provides information on the movement of sap within the tree stem.

A micro-computed tomographic image of a sugar maple branch section showing (left) a transverse two-dimensional slice of the branch section, and (right) a three-dimensional reconstruction, with a small section cut out to show the inner regions. The zoomed in region shows both sap-filled (arrowheads) and embolised vessels (white arrows). (Credit: Tenaya Driller)

Synchrotron-based microCT

At the Australian Synchrotron, we can access the imaging and medical beam line (IMBL),which produces high spatial resolution microCT images in a very short length of time (compared to bench top microCT). The large room in which the imaging is done allows us to mount a living sapling directly in front of the x-ray source, though still we only image a small region of the whole sapling.

With this technique, we are attempting to observe gas and sap microstructural changes that occur when a tree is frozen and then thawed. This freeze-thaw process occurs in nature and is believed to lead to the elevated stem pressures that allow mature maple trees to be tapped for syrup.

Synchrotron experimental set-up. Left image shows a sapling mounted using restraints used to keep the sapling fixed in place while imaging. Right image shows the same setup wrapped in polystyrene insulation. Dry ice is introduced into the polystyrene chamber to rapidly cool the sapling, and the insulation means it will unthaw very slowly. The robotic arm is used to move the entire setup in front of the beam line when imaging (Credit: Tenaya Driller).

Sections of saplings’ stems were scanned in-situ, every six-minutes throughout an induced freeze-thaw cycle. Using this novel method, we identified embolised vessels (vessels filled with gas), and showed that the status of embolised vessels changed throughout the freeze-thaw cycle.

Images of a live sapling taken on the IMBL at the Australian Synchrotron showing a sapling before freezing, during and after an induced freeze-thaw cycle. Below each is a magnified image of the regions of interest selected in white. Air-filled vessels appear as black dots while water-filled vessels appear as light grey(Source: Driller, et al., 2020).

Next Steps

In our research, we are continuing to examine these changes at high resolution and in different species, aiming to determine how the changes in microstructure may influence sap yield. We are also working on newer iterations of the cooling setup that provides a much greater degree of control over the rate at which the tree is frozen and thawed, to better mimic natural environmental temperature changes.

Sources: This blog post summarises data and findings reported in a 2020 conference paper: Driller, T., Holland, D.J. and Watson, M.J. (2020). A detailed look at the mechanisms involved in maple sap exudation. Acta Hortic. 1300, 121-130.

Plant Physiology