Imaging maple microstructures using Cryo-SEM

October 6, 2023

Our team recently visited the cryo-SEM facility at Monash University to collect images of the microstructures within sugar maple saplings.  

Scanning electron cryo-microscopy (CryoSEM) can be used to image materials with high water content. The method allows us to preserve a sample in its hydrated state, therefore providing representative images of the sample's internal structure, morphology and composition.

The sample is cryogenically fixed (i.e. rapidly cooled using liquid nitrogen) and then imaged on the scanning electron microscope's cold stage in a cryogenic chamber at around-160°C.

Our goal for the 2023 cryo-SEM work was to improve on preliminary work done by a former PhD student, aiming to observe ice formation within maple fibres after a slow freeze (i.e. one that replicates nature).

While we were able to obtain a greater number of images, we had issues with preparing surfaces for imaging.The sample preparation process is technically challenging. For our maple samples, the size of the stem sampled was limited to about 3mm width, and we discovered that freezing and cleanly cutting the samples is more art than science!

This meant we obtained very mixed results, with some images providing useful information, and others less so. Some successful examples are shown below. With refinement to the methods, the use of Cryo-SEM offers a useful technique for imaging the internal microstructures in saplings.

Cutting frozen sapling stem under liquid nitrogen in preparation for cryo-SEM imaging.
CryoSEM image of frozen maplestem. The large holes in the centre are the pith (dead, inner wood) and thesmall outer holes are the xylem in the (living) sapwood.
CryoSEM image, zoomed in on the xylem fibres.
Plant Physiology