Towards structural cell biology with superresolution microscopy
Dr. Jonas Ries, EMBL Heidelberg
Single-molecule localization based superresolution microscopy (SMLM) nowadays reaches a resolution sufficient to determine structures of protein assemblies in the cellular context. It is therefore a technique complementary to classical structural techniques such as x-ray crystallography or electron microscopy to investigate, how molecular machines are organized. In this talk, firstly I will introduce a new method for isotropic 3D SMLM and secondly I will report on our progress towards resolving a fundamental multi-protein machinery on the nanometer scale, namely the endocytotic machinery in S. cerevisiae. Supercritical angle localization microscopy (SALM) is a 3D SMLM technique based on the principle of surface-generated fluorescence. This near-field fluorescence is strongly dependent on the distance of fluorophores from the coverslip and can therefore be used to estimate their axial positions. We established a robust and simple implementation of supercritical angle fluorescence detection for single-molecule localization microscopy, calibrated it using fluorescent bead samples, validated the method with DNA origami tetrahedra, and present proof-of-principle data on biological samples. Endocytosis is a highly intricate cellular process, which in yeast involves the ordered recruitment and disassembly of around 60 proteins. Our current efforts focus on understanding the intermediate and late coat assembly preceding scission. Here, we were able to reveal subdiffraction features regarding shape and structure of endocytic coat proteins that were previously inaccessible. By visualizing many proteins pairs with dual-color superresolution microscopy, we are pursuing to obtain a comprehensive structural picture of the endocytic8 proteome.
 J. Deschamps and J. Ries, "3D superresolution microscopy by supercritical angle detection." Opt Express, vol. 22, no. 23, pp. 29081-29091, Nov. 2014.
 M. Mund, C. Kaplan and J. Ries, "Localization microscopy in yeast" in Methods Cell Biol, vol. 123, Elsevier, 2014, pp. 253-271. 3. A. Picco, M. Mund, J. Ries, F. Nédélec, and M. Kaksonen, "Visualizing the functional architecture of the endocytic machinery." eLife, vol. 4, 2015.