Parkinson's Disease (PD) is characterized by the presence so-called Lewy bodies (LBs) and Lewy neurites in affected brain regions. LBs are large intracellular aggregates, for which Spillantini et al. identified the protein alpha-synuclein (aSyn) as a major protein component. We have applied correlative light and electron microscopy (CLEM) to human post-mortem brain from Parkinson's disease patients, and investigated the structure of Lewy bodies. Contrary to expectations, most LBs that did stain strongly for phosphorylated alpha-synuclein in the light microscope, showed in the electron microscope that they consisted primarily of aggregated membranous material instead of the expected fibrillar content. In parallel, we have determined the atomic structure of in vitro generated alpha-synuclein fibrils by cryo-EM, revealing three different types of atomic structures, termed polymorphs. We here propose to further develop CLEM towards cryo-CLEM, by combining correlative fluorescence light microscopy and cryo-electron microscopy of high-pressure frozen human brain, which would avoid any chemical fixation or heavy-metal staining. This will require developing a protocol to stain human brain prior to high-pressure freezing or sectioning. We will then apply immuno-EM, conventional CLEM, and cryo-CLEM to study human post-mortem brain from patients. We will determine the molecular composition of filamentous structures found in some of the LBs, and investigate the compositions of LBs found in other brain and tissue regions. We will expand our analysis to brain tissue at earlier stages of Parkinson’s disease, and study the LB appearance in sporadic, as well as genetically triggered PD variants from patients who suffered from mutations in LRRK2 and GBA. In addition, we will seek to purify aSyn fibrils from human brain of PD patients, and determine their high-resolution structure by cryo-EM. We expect our analysis of earlier disease (Braak) stages to shed light into the chronologic development of Lewy pathology. The analysis of the ultrastructure of the post-mortem brain of PD patients who were affected by LRRK2 or GBA mutations, might give clues about the mechanism of disease development in these frequently occurring inherited forms of PD. And finally, the structure of aSyn fibrils purified from PD patient brains might allow conclusions about the mechanism of action of aSyn protein in the fibrillar vs. non-fibrillar form. Brain-derived fibril structures could then be compared with our in vitro generated structures, to identify a suitable drug target candidate among our in vitro aSyn fibril samples. In addition to PD, this research might also help in the understanding of other synucleinopathies such as Multiple System Atrophy and Dementia with Lewy Bodies.