Imaging living maple saplings during freeze-thaw cycles

September 29, 2023

Our recently team visited Melbourne to secure time on the Imaging and Medical beamline (IMBL) at the Australian synchrotron. Our goal was to use the IMBL to image the gas present within the stems of living maple saplings through a controlled cycle of slow freezing and thawing. This work improved on prior experiments, with a much larger number of samples and improved temperature control.

Experimental freeze-thaw rig

To mimic the natural free-thaw cycles in maple trees that drive sap flow, our experimental rig allows a sapling to be frozen and thawed in a controlled manner over several hours, whilst being imaged. The rig is installed on a motorised dais at the beamline, and 3D images are collected of the live maple stem, whilst controlling temperature.

During the recent work, we took images of six saplings through a freeze-thaw cycle (ca. 9 hours per cycle). Three of the saplings underwent two freeze-thaw cycles to simulate the effects of what might occur to trees over multiple days (where they would freeze each night).

LEFT: Setting up the experimental rig (in centre of image) at the IMBL. RIGHT: The sealed enclosure during testing at the University of Canterbury (NZ). The rig was a sealed polystyrene enclosure that circulated air through radiators. The radiators were fed from a chiller using ethylene glycol at -18°C. A pump controlled the flow rate from the chiller to control the enclosure temperature (there were also heaters mounted in the rig for this purpose).

Imaging Results

In earlier work on the IMBL were able to observe vessel embolisms, however the signal to noise was low making it difficult to resolve sub-pixel fibre embolisms. For these experiments, to address this noise we used a long exposure time and increased the number of individual angles scanned.

Our goal was to observe gas within vessels (approximately 30 microns in diameter) and within fibres (approximately 8 microns in diameter). Using the IMBL, we were able to achieve an effective pixel resolution of 10 microns. However, in these recent images (examples below) we do not observe any obvious vessel embolisms. Their absence is likely due to variability between saplings and differences in watering condition compared with previous experiments, rather than insufficient resolution.

Given the lack of vessel embolisms, our analysis of the images will focus on fibre embolisms. Fibres occur in large groupings, and embolised fibres appear as darker regions of the images (see arrows). Preliminary analysis suggests we can separate the embolised and non-embolised fibre regions and thus can monitor how the regions change during a freeze-thaw process. This change reflects the movement of sap during freezing which is crucial to understanding the mechanisms of sap exudation.

Example images taken on the IMBL. Red arrows point to regions that are likely gas fibres while blue arrows point to regions containing sap filled fibres and/or vessels.
3D reconstruction of sugar maple sapling stem.
Matt Watson, Matt Rennie and Jamie Robinson visited the Australian Synchrotron to collect images of the microstructures inside live maple trees.

Plant Physiology