In the first part of the project, we used a seeding model for alpha-synuclein in neurons that mimicks spreading of the disease in the brain. Samples were successfully imaged using super-resolution optical fluctuation imaging (SOFI, a type of super-resolution microscopy that analyzes higher-order statistics of fluctuations in the fluorescence emission) on our multi-plane 3D imaging platform. This allowed reconstruction of high quality images up to third order e.g. of fibrillar aggregates in neurites and in the neuronal cell bodies. Our findings are part of a publication in Nature Photonics for the ‘Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy’ that I published with PhD student A. Descloux. Later in the project, a new class of fluorescent labels for high-order SOFI was tested which opens the perspective for molecular counting in 3D. In the second part of the project, a new approach for multicolor SOFI based on the cross-cumulation between simultaneously acquired color channels was demonstrated. Furthermore, different super-resolution modalities (SMLM, SOFI, STED) were tested and used to investigate the interplay of alpha-synuclein aggregation and the neuronal cytoskeleton, with further mechanistic studies ongoing. For the third part of the project, fluorescence super-resolution was merged with white light quantitative phase imaging (QPI). QPI measurements provide information about the local thickness and refractive index of the sample at diffraction limited resolution and orders of magnitude higher imaging speed. We came up with a new way to recover phase images from bright-field z-stacks. Since we realized ultrafast 3D data acquisition by implementing an image splitting prism in the microscope detection path we could simultaneously record a z-stack of 8 planes only limited by camera speed, ideal to observe fast processes in living cells. We used this fast phase imaging to look for calcium dynamics in neurons and explored label-free detection of protein aggregates.
