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Histological tissue sections are part of everyday medical practice. With the help of dyes, they reveal tissue structures and pathological changes. However, the method requires labor-intensive steps and provides only two-dimensional insights into the tissue. An international team with significant involvement from the Helmholtz-Zentrum Hereon has now developed a non-destructive technique that combines dyes with 3D X-ray imaging. Using a new algorithm, tissue and dye can be visualized separately in 3D and quantified — opening up new possibilities for research and medicine. The research team presents its study in the journal Advanced Science.
Histology is one of the foundations of modern diagnostics. When physicians want to determine whether tissue is pathologically altered, they rely on microscopic tissue analysis: They cut the tissue into ultrathin sections, stain it with special dyes, and examine it under a light microscope. In this manner, physicians can identify whether a tumor is present and if so, what kind of tumor it is – thereby enabling informed therapeutic decisions. In addition, doctors often assess during surgery whether all altered tissue has already been removed or further intervention is required.
However, the procedure is labor-intensive: the tissue must be either frozen or fixed and embedded in wax, then cut and stained — a time-consuming process that divides the sample into many slices and tears apart its spatial context. “You cannot view the tissue in its full 3D context, for example to follow the path of blood vessels or determine where exactly the tumor ends,” explains Dominik John, first author of the study and researcher at the Hereon Institute of Materials Physics.
This is why experts are working on what is known as virtual histology — 3D X-ray imaging with micrometer-scale resolution. Unlike visible light, X-rays can penetrate samples several centimeters in thickness while providing data on the entire tissue volume. Instead of the analysis being limited to only a few selected tissue regions, any area can be examined from any desired direction. But one problem remained unsolved: X-ray images are black and white. This meant that the dyes used in classical histology to color cell nuclei or specific tissue types could not be distinguished from the surrounding tissue in X-ray images.
X-ray imaging with color information
This is where the innovative method developed by John together with an international team of researchers from Hamburg, Munich, and Melbourne comes into play. The approach combines high-resolution X-ray computed tomography with a special phase-contrast technique and a new evaluation algorithm. This algorithm simultaneously uses two distinct measurements: how strongly the tissue attenuates the X-rays and how much it refracts them. The latter becomes visible through a fine grid placed in the X-ray beam, which projects a dot pattern onto the sample. “This allows the method to compute two separate 3D images,” John explains. “One shows only the tissue, the other only the dye.”
As a demonstration, the researchers examined kidneys from mice and rats treated with the dye haematein. A lead atom was attached to the dye to enhance X-ray contrast. The team conducted their measurements at the PETRA III X-ray source at DESY in Hamburg and at the Australian Synchrotron in Melbourne. The result: the method not only shows where the dye is located — it also quantifies its amount. “We can determine the exact dye concentration for each region of the tissue sample,” says John. “This is valuable information for research.” To compare their X-ray images with conventional histological images, the team prepared tissue sections from the same sample — and found good agreement.
The method is still complex because it depends on large-scale research facilities. The experts therefore aim to make it more accessible through modern laboratory X-ray sources. “Initially, it could serve as a tool for scientific studies, for example in cancer research,” says Dominik John. “But if we can find a way to improve the resolution further, it would also become highly relevant for clinical diagnostics.” In medicine, diseased tissue could be analyzed in its full spatial context — for instance to better assess tumor spread, completeness of surgical removal, or therapeutic effects. For patients, this would be a tangible benefit: more precise diagnoses, better-informed treatment decisions, and potentially less invasive procedures.
German Engineering Materials Science Centre (GEMS)
The German Engineering Materials Science Centre (GEMS) is the central user platform of Hereon’s Institute of Materials Physics, offering a globally unique infrastructure for complementary research with photons and neutrons. The instruments that use synchrotron radiation are operated at the external site at DESY in Hamburg, while the instruments that utilize neutrons are located at the external site at the Heinz Maier-Leibnitz Center (MLZ) at the FRM II research reactor in Garching near Munich.
Cutting-edge research for a changing world
Helmholtz-Zentrum Hereon’s scientific research aims at preserving a world worth living in. To this end, around 1000 employees generate knowledge and research new technologies for greater resilience and sustainability - for the benefit of the climate, the coast and people. The path from idea to innovation leads through a continuous interplay between experimental studies, modeling and AI to digital twins that map the diverse parameters of climate and coast or human biology in the computer. This is an interdisciplinary approach that spans from the fundamental scientific understanding of complex systems to scenarios and practical applications. As an active member of national and international research networks and the Helmholtz Association, Hereon supports politics, business and society in shaping a sustainable future by transferring the expertise it has gained.
Dominik John
Scientist
Institute of Materials Physics
Tel.: +49 (0)40 8998 5301
Mail: dominik.john@hereon.de
https://doi.org/10.1002/advs.202519783
The X-ray images were acquired at the Hereon research facility P05 at DESY in Hamburg, where high-in ...
Source: Torben Fischer
Copyright: Hereon/Torben Fischer
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The X-ray images were acquired at the Hereon research facility P05 at DESY in Hamburg, where high-in ...
Source: Torben Fischer
Copyright: Hereon/Torben Fischer
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