Faculty: Hans Ulrich Dodt
It would be very helpful for the analysis of neuronal networks of the brain, if one could visualize these networks in 3 dimensions. Up to now this was only possible with limited resolution by sequential slicing and reconstruction of the brain. This time consuming attempt is easily hampered by artefacts as shrinkage and distortion induced by standard histological procedures. To overcome these problems we use a microscopy based on extreme darkfield illumination with a light sheet, once called ultramicroscopy.
This microscopy allows optical sectioning of whole mouse brains and was combined with an approach to clear fixed neuronal tissue: Mouse brains are made completely transparent and by illuminating the brains with blue light (λ = 488 nm), neurons labelled with GFP are visualized by fluorescence. This way we can detect single neurons in hippocampi inside whole brains as it was already shown for spinal cords of mice (Figure). By surface rendering the shape and position of hippocampi relative to the brain surface can be depicted. In complete excised hippocampi subcellular resolution is obtained by 3D reconstruction from several hundred optical sections. The dendritic trees of CA1 hippocampal neurons with dendrites and dendritic spines can be visualized by labelling proteins in transgenic mice with genetically encoded fluorescent markers. Using these markers our approach will represent a high-throughput screening method for protein expression in 3 D.
However resolution of the images is still limited by diffraction. It would be advantageous for many questions of synapse formation and neuronal plasticity if a resolution well below the diffraction limit could be reached. We propose to use a technique called dSTORM (collaboration with G. Schütz). We want to apply this approach to every optical section in ultramicroscopy. In addition it would be very interesting to have multicolour reconstructions with different colours of pre- and postsynaptic elements. Thus changes in spine heads in relation to presynaptic elements could be studied. The dSTORM superresolution images will be correlated with global images recorded on intact, cleared, and fluorescently stained brain with our light-sheet laser scanning microscopy approach (1).
1. Ertürk, A., Becker, K., Jahrling, N., Mauch, C.P., Hojer, C.D., Egen, J.G., Hellal, F., Bradke, F., Sheng, M., and H.U. Dodt. 2012. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat Protoc 7:, 1983-1995.
- International collaborations are documented for Dr. Frank Schnorrer (Max-Planck-Institute of Biochemistry, Munich), Prof. Matthias Jucker (Hertie Institute, University Tübingen), Prof. Frank Bradke (DZNE, Helmholtz Gesellschaft, Bonn), Prof. Manfred Frasch (Chair of Developmental Biology, University Erlangen) and Prof. Henry Markram (EPFL, Lausanne).
- The PALM technique shall be implemented in collaboration with G. Schütz (TU Vienna) who has documented experience in the field. If images are obtained in different colours the single images do not completely overlap due to chromatic aberration in the clearing solution.
- Image superposition is essential. This can be done by special warping techniques developed in computer science. This approach is routine for R. Sablatnig (TU Vienna).