Tissue Preparation

Tissue Preparation

The foundations of our research are established long before the imaging process begins. First, brain specimens must be processed to preserve their delicate anatomical structures and make them accessible for high-resolution analysis. This process requires specialised equipment, controlled temperatures, and great manual precision.

The specimens are initially placed in a special embedding medium to stabilise the delicate structures. They are then cut into serial sections in the micrometre scale using a large-section cryostat at −50 °C. Each section is then frozen at −80 °C to preserve the tissue structure in the long term. In the case of a human brain, this can result in around 3,000 individual sections.

For further analysis, selected sections are thawed, covered with glycerine and weighed down overnight. The aim is to achieve a preparation that is as uniform and bubble-free as possible. This step is particularly challenging with large-area sections: carefully removing the tiniest air bubbles can take up to two hours per specimen and requires great care to avoid damaging the sensitive tissue.

Working with large-format whole-brain sections poses an additional challenge. Unlike smaller tissue samples or individual brain regions, these specimens provide a view of interconnected structures within a broader anatomical context. Processing them requires technical precision, expertise and patience.

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Staining with cresyl violet

After the 3D-PLI measurement has been taken, selected tissue sections are processed again to obtain additional histological information. To achieve this, the sections are coverslipped and prepared for various staining procedures. One commonly used method is staining with cresyl violet.

This stain binds to neuronal structures, particularly RNA-rich regions within the cell body, making neuronal somata clearly visible. This provides a clear depiction of the cytoarchitectonic structures of brain tissue, i.e. the distribution, density and organisation of nerve cells in different brain regions.

Thus, cresyl violet staining complements the previously acquired 3D-PLI data at the cellular level. Together, these two methods enable a more comprehensive characterisation of the brain’s structural organisation.

Silver staining

Another method used is silver staining, which is particularly employed to visualize myelinated nerve fibres. Myelin is a lipid‑rich insulating layer that surrounds axons in the brain, and plays a crucial role in rapid signal conduction.

Through silver impregnation, these myelin structures are selectively stained and made visible in the tissue section. Depending on the staining protocol used, the fibre pathways appear as fine, high‑contrast structures that clearly highlight the organization of the nerve fibre tracts in the brain.

This method complements other techniques such as cresyl violet staining, as it emphasizes the fibre architecture rather than the cell bodies. This provides a detailed picture of the structural organization of the brain at different levels, from cellular architecture to the course of fibre pathways.

Staining with Luxol Fast Blue

Luxol Fast Blue can be used to stain the sections after 3D-PLI analysis, enabling the visualisation of the characteristics of the myelin surrounding the nerve fibres. This clearly highlights the myelin structures, enabling a detailed assessment of the degree of myelination, regional differences and potential demyelinating changes. At the same time, Luxol Fast Blue improves understanding of specific patterns in the 3D-PLI polarization signal: differences in retardation or fibre organisation can be directly correlated with the actual distribution of myelin. This significantly improves the interpretation of 3D-PLI data.

Combining both methods provides a more comprehensive picture of the structural and microstructural properties of the same tissue because the optical information from 3D-PLI can be directly linked to the biochemical and morphological characteristics of myelin.

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