Rudolf Virchow fundamentally changed medicine when he formulated his cell theory of disease in the 19th century: diseases do not arise inexplicably within the organism, but rather in specific cells and tissues. To this day, pathology – the study of disease processes – is essentially based on the time-consuming examination of thin tissue sections, which are stained and then viewed under a microscope.
Now an international research team at the Paul Scherrer Institute PSI has managed to overcome this two-dimensional limitation. Using high-resolution micro-computed tomography (µCT) and artificial intelligence, a group led by physicist Goran Lovric from the PSI Center for Photon Science generated virtual stains of tissue samples, so-called histological stains. This could potentially eliminate the need to prepare and stain ultrathin, delicate sections. "We have shown for the first time that a CT-based virtual stain can deliver results similar to conventional laboratory histology," Lovric explains. "This could open up a wealth of clinical and scientific applications."
Familiar colour markers of histology
The researchers combined high-resolution phase-contrast micro-CT (PCµCT) with machine learning methods. The platform is called VISTACT – short for virtual staining of micro-computed tomography. While conventional computed tomography primarily measures differences in X-ray density, phase-contrast micro-CT utilises additional X-ray information, thereby achieving significantly better visualisation of soft tissue. This allows three-dimensional visualisation of fine anatomical structures on the micrometre scale – so far, however, only in greyscale. In pathology, however, specialists are trained to interpret the typical colour contrasts of conventional histological stains: cell nuclei appear blue-violet, collagen pink, and elastic fibres dark. Many of these visual reference points are lost in greyscale CT datasets.
"We therefore wanted to carry over the familiar colour world of histology to three-dimensional CT data," explains Lovric. To achieve this, the researchers trained a specialised AI model using pairs of real histological sections and their corresponding CT scans. In this way the AI model learned which microscopic patterns typically receive which staining. It then was able to virtually stain new CT data – essentially an automatic translation between two image worlds.
More precise localisation
One crucial technical step was precise mapping of the images. Histological sections are only a few micrometres thick and can easily become distorted during sectioning or mounting. In addition, it is essential to determine exactly where each section is located within the three-dimensional CT dataset. Lovric's research group developed a multi-stage process that automatically identifies the corresponding layer and compares it with the histology data. According to the researchers, this spatial mapping is significantly more precise than previous standard methods.
To carry out the virtual staining, researchers used a so-called conditional generative adversarial network – a specialised AI model for image-to-image translation. With greyscale images from micro-CT scans as input, the model generated virtual histological specimens. Remarkably, the AI produced not merely coarse colour areas but rather plausibly differentiated tissue components of various types: blood in the fine vessels appeared yellowish, collagen structures pink, and surfaces in the lungs grey to violet.
Lung tissue test provides proof of concept
The researchers tested their new method on lung tissue taken from individuals with pulmonary hypertension. This condition involves pathological remodelling of the pulmonary vessels. "We were able to map the altered vascular regions in three dimensions," says Cristina Almagro-Pérez. She is the first author of the new publication and worked in Goran Lovric's group during her master's thesis. She is now doing research in the USA.
The new technique can be automated and can work significantly faster than the current method. However, it is not yet ready for routine use in hospitals: the necessary phase-contrast imaging was performed at the TOMCAT beamline of the Swiss Light Source SLS, one of the large research facilities at PSI. The resulting volumes of data were enormous, and the resolution was often insufficient to depict individual cell nuclei reliably.
Furthermore, virtual histology remains a statistical reconstruction: the AI platform does not generate actual histological information, but rather plausible predictions based on the training data. Almagro-Pérez and Lovric emphasise that the procedure has not yet reached routine diagnostic quality. However, the "proof of concept" has been established, and the method is, in principle, applicable to the examination of various diseases. Particularly in examining tumours, vascular lesions, or complex tissue architectures, this form of non-destructive 3-D pathology has the potential to accelerate research into disease biomarkers and thus to open up new diagnostic perspectives in the long term.
More than 150 years after the advent of Virchow's cellular pathology, histology might again be on the verge of a fundamental transformation.
Text: Werner Siefer
About PSI
The Paul Scherrer Institute PSI develops, builds and operates large, complex research facilities and makes them available to the national and international research community. The institute's own key research priorities are in the fields of future technologies, energy and climate, health innovation and fundamentals of nature. PSI is committed to the training of future generations. Therefore about one quarter of our staff are post-docs, post-graduates or apprentices. Altogether PSI employs 2300 people, thus being the largest research institute in Switzerland. The annual budget amounts to approximately CHF 450 million. PSI is part of the ETH Domain, with the other members being the two Swiss Federal Institutes of Technology, ETH Zurich and EPFL Lausanne, as well as Eawag (Swiss Federal Institute of Aquatic Science and Technology), Empa (Swiss Federal Laboratories for Materials Science and Technology) and WSL (Swiss Federal Institute for Forest, Snow and Landscape Research).
