Modern biomedical research depends on the ability to see what is happening inside biological tissue. Among the available imaging techniques, X-ray-based micro-computed tomography, or micro-CT, has become a powerful tool for producing high-resolution three-dimensional images of small samples.
For hard, mineralised tissues such as bone and teeth, this is relatively straightforward. These structures are dense, absorb X-rays efficiently, and therefore appear clearly in micro-CT images. Soft tissues-including blood vessels, cells, and connective tissue are far more challenging. They interact only weakly with X-rays and are therefore difficult to visualise in three dimensions.
In his PhD research, Torben Hildebrand set out to address this challenge by refining contrast-enhanced micro-CT, focusing on how soft tissue can be made visible without destroying the sample.
A technical challenge at its core
Rather than being driven by a single biological question, the work is rooted in a more fundamental technical problem: what determines what we can and cannot see in micro-CT images.
"Revealing biological ultrastructure is not primarily a biological challenge," Hildebrand explains. "It is fundamentally a question of physics and engineering. What we see depends on how tissue interacts with X-rays and how that information is captured."
In other words, the visibility of biological structures is limited not by their complexity, but by how well imaging techniques are adapted to their physical properties.
Why micro-CT offers a different perspective
Micro-CT differs from optical imaging methods such as confocal microscopy in an important way. While optical techniques are limited by light scattering and shallow penetration depth, X-rays can penetrate much deeper into biological samples.
"With micro-CT, we can study biological samples from a completely different perspective," says Hildebrand. "We are not limited by how far visible light can travel into tissue, which opens new possibilities for three-dimensional analysis."
This makes micro-CT particularly useful for large or optically complex samples that are difficult or impossible to image with conventional microscopy.
The problem with soft tissue
Despite its advantages, micro-CT has a significant limitation. Soft tissues absorb X-rays very poorly and therefore produce little contrast in standard scans.
"Soft tissues are almost invisible to X-rays," Hildebrand says. "They don't cast a clear shadow. That is why contrast enhancement is so crucial in absorption-based micro-CT."
For this reason, micro-CT has traditionally been used mainly for mineralized tissues, while soft tissue imaging has relied on alternative methods.
Making soft tissue visible with contrast agents
Hildebrand's solution focuses on sample preparation rather than scanner design. By immersing samples in carefully selected contrast agents, soft tissue components can bind substances that strongly attenuate X-rays.
"You can think of contrast agents as highlighters for tissue," he explains. "They don't change the structure itself, but they make specific components visible by increasing X-ray contrast."
Different contrast agents bind to different tissue components, such as cells, blood vessels, or particular molecular structures. A central part of the work has therefore been to determine which agents are best suited for specific research questions, and how preparation protocols influence image quality.
"The key question is not whether contrast agents exist-they do," Hildebrand says. "It is how to choose the right one and prepare the sample in the right way for the structure you want to see."
Dental tissue as a model system
Much of the research is based on dental samples, which often contain both hard and soft tissues in close proximity. Teeth therefore provide an ideal model system, combining highly mineralized structures with delicate soft tissues such as pulp and vascular networks.
In typical experiments, intact teeth were sometimes partially decalcified to reduce the overwhelming contrast from hard tissue. The samples were then immersed in contrast agents, such as iodine-based solutions, which selectively bind to soft tissue components, including blood cells. This made it possible to visualize internal soft tissue structures in three dimensions while keeping the sample intact.
Virtual histology in three dimensions
The resulting image datasets enable what is known as virtual histology, a non-destructive alternative to traditional histological methods. Conventional histology requires samples to be cut into thin sections, stained, and examined under a microscope. While highly detailed, this process is irreversible and limited to two dimensions.
"Virtual histology allows us to explore tissue in three dimensions without destroying the sample," Hildebrand explains. "We can create digital slices in any direction we want."
This reduces the number of samples needed and allows complex structures to be examined from multiple perspectives. Virtual histology can also guide conventional histology.
"It fits naturally into existing workflows," he says. "We can first see the whole structure and then decide where destructive analysis is most informative."
Bringing synchrotron-level imaging into the laboratory
Another key aspect of the dissertation is the comparison between laboratory-based contrast-enhanced micro-CT and imaging performed at synchrotron radiation facilities. Synchrotrons provide extremely intense and well-controlled X-ray beams, but access is limited and highly competitive.
"With optimized contrast-enhanced micro-CT, we can achieve image quality that comes surprisingly close to synchrotron imaging, directly in the lab," Hildebrand says.
This could significantly reduce dependence on large-scale facilities and make advanced soft-tissue imaging more widely available.
Applications beyond dentistry
Although rooted in dental research, the methods developed have much broader relevance. They can be applied to biomaterials, tissue-engineered constructs, and organoids, lab-grown, three-dimensional structures derived from stem cells.
"These techniques are not limited to dentistry," Hildebrand emphasizes. "They are highly relevant for biomaterials, tissue engineering, and organoids, where structures are often too complex for traditional microscopy."
A tool for non-destructive 3D biology
The work demonstrates that progress in biomedical imaging depends not only on improved hardware, but also on smarter use of physics, chemistry, and sample preparation. By optimizing contrast-enhanced micro-CT, it becomes possible to explore biological complexity in three dimensions without destroying the sample.
As imaging systems, computational power, and AI-based analysis continue to advance, these methods are likely to become an increasingly important part of biomedical and materials research.
