3D X-ray microscopy pioneered for plant imaging

Researchers at the Donald Danforth Plant Science Center in the US have used X-ray microscope technology to image plant cells, whole tissues and even organs at unprecedented depths with cellular resolution.

Their work, published in the journal Plant Physiology, should enable plant scientists globally to study above- and below-ground traits at supposedly unprecedented clarity.

Measuring plant phenotypes, a term used to describe the observable characteristics of an organism, is a critical aspect of studying and improving economically important crops. Phenotypes central to the breeding process include traits like kernel number in corn, seed size in wheat or fruit colour in grape. These features are visible to the naked human eye but are in fact driven by microscopic molecular and cellular processes in the plant.

“Plants are multiscale,” explained Dr Christopher Topp, who co-led the new research alongside scientist Keith Duncan. “An ear of corn starts off as a microscopic group of cells called a meristem. Meristem cells will eventually form all the visible parts of the corn plant through division and growth.”

Using three-dimensional (3D) imaging is a recent innovation in the plant biology sector to capture phenotypes on the ‘whole-plant’ scale: from minuscule cells and organelles in the roots, up to the leaves and flowers. However, current 3D imaging processes are limited by time-consuming sample preparation and by imaging depth, usually reaching only a few layers of cells within a plant tissue.

The use of 3D X-ray microscopy (XRM) technology allowed Dr Topp and Duncan to relate the developmental microstructure of the plant, such as meristem cells, to visible traits as they mature, such as leaves and flowers; in other words, 3D XRM provides cellular-level resolution of entire plant organs and tissues. In addition, the methodology can image below-ground structures at exceptional resolution, including roots, fungi and other microbes.

“Plant roots drive a lot of important biological processes; they feed microbes in the soil, and in return the plants get phosphorus and nitrogen,” Dr Topp said. “We know the interaction between roots and microbes is important because it was a primary source of phosphorus and nitrogen before we invented chemical fertilisers.”

Our dependency on chemical fertilisers in standard agricultural practices has, in turn, made major contributions to global climate change. Therefore, a critical component of the sustainable agriculture movement includes reducing chemical inputs and instead fostering natural interactions between roots and microbes below ground.

“We haven’t had the tools to understand these interactions until recently,” Dr Topp said. “3D XRM can help unlock the potential of re-establishing these natural alliances in our agriculture systems.”

Furthermore, 3D XRM methodology is claimed to be unique compared to other imaging approaches in plant biology because of its ability to yield essentially perfect 3D clarity of plant structure. Other common methods, such as photon-based tomography, are limited by shallow imaging depths and are optimised in a select few species of plants. In contrast, by using 3D XRM, Dr Topp and Duncan are able to image thick tissues that are recalcitrant to typical, optical methods in a whole host of economically important crops, including corn, foxtail millet, soybean, teff and grape.

A major goal of the team’s paper was to establish a reproducible protocol for other plant scientists interested in 3D XRM imaging. To do so, Duncan spent a lot of time preparing samples to optimise the contrast between the plant and its background. X-ray imaging works through differential absorption, where dense material (like minerals in the soil) absorbs more X-rays and shows up darker on an image. However, biological matter like plant tissue has low X-ray absorption, and the team was at risk of completely washing out the material they were interested in imaging.

“Solving that problem for one kind of sample — like a root tip — is one thing,” Dr Topp said, “but the idea of the paper was to give plant scientists working on a variety of relevant plant tissues and species the access to these methods. We want to broadly apply 3D XRM to plant systems above and below ground.” As such, the team’s published methodologies should greatly advance the number of plant species and the types of plant tissues that can be imaged at nearly perfect resolution.

Next on the horizon is to image 3D structures of fungal networks in the soil. Part of that work includes improving machine learning approaches, such that a computer is trained to recognise what within an image is a root, soil or spore. The team’s work should thus continue to develop new technological approaches to improve our multiscale understanding of the whole plant, from the microscopic to the visible.

Image caption: Volume rendering of a single developing soybean flower shows the relationship of pollen-filled anthers, ovules and stigmatic surface to one another in 3D space. Image courtesy of the study authors.

Please follow us and share on Twitter and Facebook. You can also subscribe for FREE to our weekly newsletters and bimonthly magazine.