Development of a new tissue preparation protocol for the study of whole mouse brain connectivity

Understanding brain function and dysfunction requires detailed mapping of neural connections at the single-axon level across entire circuits. A common challenge is balancing the need to image large areas with the resolution to resolve tiny axons, which are smaller than light's diffraction limit. This thesis addresses this by developing an integrated method combining tissue clearing, expansion microscopy (ExM), and advanced light-sheet imaging, enabling super-resolution connectomics of the whole mouse brain. We introduce a novel technique that sequentially employs the iDISCO+ clearing protocol and a specially tailored ExM hydrogel embedding. Key optimisations involve a specific hydrogel monomer formulation, a permeabilisation step for uniform expansion, and a potent signal amplification strategy using the FlexAble 2.0 labelling kit from Proteintech. This integrated approach produces robust, fully transparent samples with about 4-fold linear expansion, effectively surpassing the diffraction limit. It is complemented by a custom-built light-sheet microscope designed for efficient imaging of these large, expanded samples. The final protocol enables clear visualisation and tracing of sparse, long-range axonal projections throughout the entire uncut mouse brain. It overcomes major challenges such as sample fragility, signal dilution, and complex processing, creating a reliable, user-friendly platform for high-resolution whole-brain connectomics. This method bridges the gap between mesoscale mapping and nanoscale detail, supporting the study of neural circuits in health and disease.