Periodic Reporting for period 1 - funcSOFI-PD (Quantitative super-resolution optical fluctuation and phase microscopy for structural and functional imaging of Parkinson's disease)
Período documentado: 2017-04-01 hasta 2019-03-31
One of the big challenges today and in the future is the consequences of a population that is getting older.
In 2030, 25% of the population in Europe are expected to be over 65, thus it is more likely that people develop neurodegenerative age-linked diseases like Alzheimer’s and Parkinson’s.
Those diseases have a huge impact on quality of life and to date, therapies can only treat the symptoms. Oligomerization and
aggregation of the protein α-synuclein is a hallmark of Parkinson’s disease. However, the underlying molecular mechanism
of cellular dysfunction due to toxic forms of this protein remains unclear. The overall objective of this project was to investigate alpha-synuclein (α-syn) aggregation
in Parkinson’s disease compromised neurons using advanced microscopy techniques. I suggested to develop and apply multicolour 3D quantitative fluorescence
super-resolution optical fluctuation imaging (SOFI) and label-free phase microscopy in a multiplane microscope setup.
The goal was to use this new imaging concept(s) (1) to follow α-syn aggregation over time at the cell membrane and in the cytosol and (2) to uncover the induced
cytoskeleton alterations in mouse primary hippocampal neurons. Ultimately, the molecule-specific readout based on SOFI was be complemented by (3) label-free,
quantitative phase imaging of dynamics and overall cell morphology at extremely high imaging speeds.
I am a biophysicist with a specialization in single molecule spectroscopy and in my PhD I worked on using photon statistics for molecular counting.
This fellowship has expanded my quantitative microscopy expertise to 3D imaging
while enlarging my biological scope to neurodegenerative diseases. I could successfully develop novel tools for advanced microscopy and
apply them to monitor the interplay of cytoskeleton and protein aggregation in Parkinson's disease. I could also extend my personal competences by managing
the project, following complementary skills courses, and organizing public outreach activities (e.g. study week for high-school students). While performing
interdisciplinary research on neurodegenerative disease, I could strengthen my research profile to further
enhance the European research landscape.
In 2030, 25% of the population in Europe are expected to be over 65, thus it is more likely that people develop neurodegenerative age-linked diseases like Alzheimer’s and Parkinson’s.
Those diseases have a huge impact on quality of life and to date, therapies can only treat the symptoms. Oligomerization and
aggregation of the protein α-synuclein is a hallmark of Parkinson’s disease. However, the underlying molecular mechanism
of cellular dysfunction due to toxic forms of this protein remains unclear. The overall objective of this project was to investigate alpha-synuclein (α-syn) aggregation
in Parkinson’s disease compromised neurons using advanced microscopy techniques. I suggested to develop and apply multicolour 3D quantitative fluorescence
super-resolution optical fluctuation imaging (SOFI) and label-free phase microscopy in a multiplane microscope setup.
The goal was to use this new imaging concept(s) (1) to follow α-syn aggregation over time at the cell membrane and in the cytosol and (2) to uncover the induced
cytoskeleton alterations in mouse primary hippocampal neurons. Ultimately, the molecule-specific readout based on SOFI was be complemented by (3) label-free,
quantitative phase imaging of dynamics and overall cell morphology at extremely high imaging speeds.
I am a biophysicist with a specialization in single molecule spectroscopy and in my PhD I worked on using photon statistics for molecular counting.
This fellowship has expanded my quantitative microscopy expertise to 3D imaging
while enlarging my biological scope to neurodegenerative diseases. I could successfully develop novel tools for advanced microscopy and
apply them to monitor the interplay of cytoskeleton and protein aggregation in Parkinson's disease. I could also extend my personal competences by managing
the project, following complementary skills courses, and organizing public outreach activities (e.g. study week for high-school students). While performing
interdisciplinary research on neurodegenerative disease, I could strengthen my research profile to further
enhance the European research landscape.
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.
During the project, we developed a number of new microscopy tools (multi-plane microscope for ultrafast 3D data acquisition, a new approach for quantitative phase imaging using brightfield image stacks and Fourier filtering, a new approach for robust and simple-to-use multicolor SOFI imaging). They were in this project used to investigate Parkinson's disease, in the meantime our advanced microscopy could already be extended to Huntington's disease imaging. In principle, these tools can be used for investigation of other diseases where super-resolution and label-free imaging promise to help uncover new biology. In addition, the project resulted in a new way to estimate the resolution of images. This is extremely important to optimize microscope adjustment and acquisition and post-processing of images and could be crucial for setting up automated multimodal microscopes. Ongoing experiments promise to reveal the connection between alpha-synuclein aggregation and the neuronal cytoskeleton, adding a piece to the puzzle of molecular mechanisms behind neurodegerative disease.