Exploring the 3D architecture of brain tissue using digital holographic microscopy

To understand the complexity of the brain, it is necessary to study its microscopic neuronal architecture and densely packed nerve fibre networks. Techniques based on histological sectioning and staining are often used for this purpose. But they can obscure or destroy valuable information and often require extensive computational post-processing for analyzing histological images. Digital holographic microscopy (DHM) enables phase and volumetric imaging. It is a promising alternative to imaging transparent biological samples with minimal preparation and high resolution. The presented study introduces DHM to image the amplitude and phase of rat brain tissue using the double-sideband (DSB) filtering technique, while reducing phase artifacts through the incorporation of unfiltered holograms into the reconstruction formalism. Combining the reconstructed complex-valued hologram with digital processing and digitally synthesized dark-field and phase contrast filtering – including the computational evaluation of light propagation and autofocusing criteria – enhances two-dimensional structural visualisation and reveals volumetric features. This approach successfully resolves the three-dimensional arrangement of crossing fibre bundles from a single acquired hologram through indirect, depth-resolved localization, which is challenging in many imaging applications. Finally, the technique is shown to be scalable, enabling full brain section scanning while supporting a compact, intrinsically multimodal imaging setup.