Posted by What if decades-old human tissue archives could be transformed from thin, two-dimensional histological sections into detailed three-dimensional maps of cells, vessels, nerves and tissue architecture?
A new study published in Science Advances takes an important step in that direction. The researchers introduce a method called aDISCO: archival 3D Imaging of Solvent-Cleared Organs, a tissue-clearing method designed specifically for large, long-term stored formalin-fixed, paraffin-embedded (FFPE) human tissue blocks.
And, interestingly from a cyclodextrin perspective, the protocol contains methyl-β-cyclodextrin as part of its intensive tissue-permeabilization strategy.
Why is 3D histology so difficult?
FFPE tissue archives represent an enormous resource for biomedical research. Human surgical and autopsy specimens can remain preserved for many years, creating collections containing tissues from both healthy individuals and patients with rare diseases.
The problem is that conventional histology examines these samples mainly as thin sections—typically only a few micrometers thick. This means that researchers see a very small fraction of the original specimen at any one time.
Three-dimensional microscopy could overcome this limitation, but intact human tissue is not naturally transparent. Lipids, proteins, pigments, and other structures scatter and absorb light. In addition, formalin fixation creates extensive cross-linking, while paraffin embedding and long-term storage can mask epitopes and make antibody penetration difficult.
The challenge becomes particularly substantial for dense, lipid-rich tissues such as brain white matter and liver.
The new aDISCO method was developed to make large archival FFPE human tissue blocks transparent while retaining the ability to immunolabel them.
The approach combines several strategies. The researchers use repeated treatment with tetrahydrofuran (THF) and dichloromethane (DCM) for aggressive delipidation, suppress autofluorescence using acidic copper sulfate, and gradually increase antibody concentrations to promote antibody penetration throughout the tissue.
The result is remarkable: the method was tested on 11 different human organs and with 20 commonly used antibodies. It enabled imaging throughout entire archival tissue blocks, including samples that had been stored for up to 15 years.
One of the most intriguing details in the experimental protocol is the use of methyl-β-cyclodextrin during the strong permeabilization and decolorization stage.
After initial treatment with Triton X-100, the tissue was exposed to a solution containing urea, tetrakis(2-hydroxypropyl)ethylenediamine and Triton X-100. This was followed by a three-day incubation containing DMSO, glycine, Triton X-100, and 0.5 mM methyl-β-cyclodextrin.
This is a particularly interesting application of cyclodextrin chemistry. In the context of aDISCO, the authors incorporate it into an already highly intensive permeabilization procedure.
What can aDISCO reveal?
The researchers demonstrated the method in the human brain, spinal cord, peripheral nerves, skin, muscle, heart, kidney, liver, spleen, colon, and lung.
The resulting images reveal structures that are difficult – or sometimes impossible – to appreciate in conventional two-dimensional sections: branching neuronal processes, vascular networks, muscle architecture, renal glomeruli and tubules, bile ducts, pulmonary structures, peripheral nerve fibers and intestinal crypts.
The method reached imaging depths of several millimeters. In one archival lung sample, the maximum imaging depth was 6.5 mm, corresponding to the entire thickness of the cleared specimen.
Importantly, the researchers could also return cleared samples to conventional histology by reparaffinizing them, providing a bridge between emerging 3D approaches and established diagnostic techniques.
Reference
Reuss, A. M., Groos, D., Cerisoli, M., Nordberg, L., Frick, L., Voigt, F. F., Vladimirov, N., Bethge, P., Reimann, R., Helmchen, F., Rupprecht, P., & Aguzzi, A. (2026). aDISCO: A clearing method to enable 3D microscopy of large archival paraffin-embedded human tissue blocks. Science Advances, 12(33), eaef9926. https://doi.org/10.1126/sciadv.aef9926.