Abstract / Summary
To achieve 3D reconstructions of large biological objects such as whole cells with ultrastructural details a generalized serial thinning, aka sectioning is necessary for any super-resolution light or electron microscopy (EM) imaging modality. While other groups focus on destructive methods such as focused ion beam milling or the serial 3View® blockface imaging we aim for a quantitative non-destructive, correlative and hierarchical sectioning and imaging workflow.
As starting point we analysed the parameters influencing the sectioning process itself and the necessary handling of thousands of sections while reproducibly setting them in usable arrays for light microscopy and Scanning EM. We defined a set of crucial properties for sample preparation and the sectioning equipment and devised a new tool for section handling. With this new equipment it was possible for the first time to handle serial section ribbons on glass cover slips for LM and EM imaging (now in collaboration with Carl Zeiss Microscopy, Oberkochen). Also important is the finding that conventional optical techniques for thickness determination (such as interference colours) of the individual sections are not accurate enough for a quantitative interpretation of the 3D reconstruction e.g. for analysis of membrane topology.
With the results obtained in the past first year of our collaboration we managed to implement the so called hierarchical Array Tomography, a combined LM/EM workflow combining different imaging modalities at different length and resolution scales. The data and new schemes have been presented at international conferences and have also been published. Further we used the results of this project to support the – successful - application for a larger new BMBF project.