Development of Techniques for Neuroimaging at Nanoscale

Abstract

A wide range of diseases is associated with alterations of the brain morphology. Multiscale three-dimensional visualization of continuous volumes of unstained brain tissue is important to gain insight to pathologically relevant processes of neurological diseases. Many pathological processes in neurodegenerative disorders affect myelinated axons, which are a critical part of the neuronal circuitry. Cryo-ptychographic X-ray computed tomography (cryo-PXCT) in the multi-keV energy range is an emerging technology, which provides phase contrast at high sensitivity, allowing label-free and non-destructive three-dimensional imaging of relatively large continuous volumes of tissue. The first part of this thesis reports a pipeline for cryo-PXCT of hydrated and unstained biological human brain tissue of volumes beyond what is typical for X-ray imaging, combined with complementary methods. Four samples of a Parkinson’s diseased human brain and five control samples from a non-diseased human brain were imaged using cryo-PXCT. In both cases, specific features were distinguished, such as neuromelanin-containing neurons, lipid and melanic pigments, blood vessels and red blood cells, and nuclei of other brain cells. In the diseased samples, several swellings containing dense granular material were observed, which were resembling clustered vesicles between the myelin sheaths arising from the cytoplasm of the parent oligodendrocyte, rather than the axoplasm. The pathological relevance of such swollen axons in adjacent tissue sections was further investigated by immunofluorescence microscopy for phosphorylated alpha-synuclein. Since cryo-PXCT is non-destructive, the large dataset volumes were used to guide further investigation of such swollen axons by correlative electron microscopy and immunogold labeling post X-ray imaging, a possibility demonstrated for the first time. Interestingly, protein antigenicity and ultrastructure of the tissue were preserved after the X-ray measurement. As many pathological processes in neurodegeneration affect myelinated axons, this work sets an unprecedented foundation for studies addressing axonal integrity and disease-related changes in unstained brain tissues. iv The extreme complexity of the human brain prevents performing precise analysis of specific components of neuronal networks, for example, of the individual neurons’ interactions in specified circuits. The second part of this thesis reports on the development of a novel compartmentalized neuronal co-culture platform allowing simulation of parts of neuronal networks with higher variable control. Polymer microstructures were fabricated on top of sapphire discs, which can direct the outgrowth of neurites originating from two distinct groups of neurons growing in different compartments. This was demonstrated using two populations of neurons expressing either EGFP or mCherry, which also facilitates the analysis of the specific interactions between two sets of cells. The design of device permits direct observation of neuritic processes within microchannels by optical microscopy with high spatial and temporal resolution to investigate the response of neurites projections to the guidance cues. Furthermore, the cell culture platform is compatible with high-pressure freezing. This allows cryo-preservation of reconstructed neuronal networks at near-native vitreous state for high resolution analysis by electron microscopy. Following freeze substitution, it is possible to process the resin-embedded neuronal networks via conventional methods for ultrastructural imaging via electron microscopy. Several key features of the embedded neuronal networks, including mitochondria, synaptic vesicles, axonal terminals, microtubules, with well-preserved ultrastructures were observed at high resolution using Focused Ion Beam – Scanning Electron Microscopy (FIB-SEM) and Serial sectioning - Transmission Electron Microscopy (TEM). These results demonstrate the compatibility of the platforms with both optical microscopy and electron microscopy. For future studies, the presented platform can be extended to disease models to allow investigating neurodegenerative processes at the nanoscale as well as pharmacological testing and drug screening. In summary, the techniques for neuroimaging reported in this thesis can be considered as essential tools for investigating the ultrastructure of sub-cellular processes within neuronal networks in normal and disease conditions.