Figure 1: Dual-colour super-resolution images of Cdc11 (red) and the cell-wall marker ConA (green) show the formation and disassembly of the Cdc11 ring.

Figure 1: Dual-colour super-resolution images of Cdc11 (red) and the cell-wall marker ConA (green) show the formation and disassembly of the Cdc11 ring.

Figure 2: Actin in yeast. Yeast expressing Abp1- mMaple imaged by localisation microscopy. Five fixed example sites at different endocytic time points.

Figure 2: Actin in yeast. Yeast expressing Abp1-mMaple imaged by localisation microscopy. Five fixed example sites at different  endocytic time points.

The Ries group develops cutting-edge superresolution microscopy methods to determine structures of multi-protein assemblies in the cellular context.

Previous and current research

The resolution of optical microscopy is limited by diffraction to about 200 nm, which is much larger than the relevant length-scales in cell biology, defined for example by the size of organelles or multi-molecular complexes. Single-molecule localisation-based super-resolution microscopy (localisation microscopy) overcomes this limit by stochastic activation and subsequent localisation of individual fluorophores, reaching a resolution in the 10 nm range.

In the past, we worked on improved labelling schemes for super-resolution microscopy. We established nanobodies as tiny, high-affinity labels, which allow any GFP-tagged protein to be used directly for localisation microscopy. As an alternative to using photo-switchable fluorophores, we introduced binding-activated localisation microscopy (BALM), which employs fluorescence enhancement of fluorogenic dyes upon binding to target structures for superresolution microscopy, to study DNA structures and alpha-synuclein amyloids and demonstrated a superb labelling density combined with a very high resolution.

Currently, one focus of the group is the development of new tools for superresolution microscopy. In one project, we are establishing a robust and simple method for isotropic 3D resolution based on supercritical angle fluorescence detection. Furthermore, we aim at measuring absolute copy numbers of proteins in large complexes by using artificial brightness standards. Combining localisation microscopy with electron microscopy in a correlative approach allows us to add molecular specificity to the ultrastructure. Single-molecule microscopy with light-sheet illumination reduces the background in thick samples.

A second focus of our work is the application of our newly developed tools to address cell biological questions. Here, we are aiming to chart a comprehensive superresolved structural picture of the endocytic machinery as well as of the kinetochore complex in S. Cerevisiae. This has been impossible so far with conventional techniques due to their complexity and small size. Furthermore, we are investigating intracellular aggregation of Parkinsons’ alpha-synuclein.

We are also developing novel data analysis tools and an open-source software platform for super-resolution microscopy. This will allow us to extract information about protein structures from super-resolution microscopy data.

Future projects and goals

Our vision is to establish super-resolution microscopy as a tool for structural cell biology in situ to bridge the methodological gap that currently exists between cell biology and structural biology techniques. We aim to push its limits on all fronts to establish a technique which combines nanometre 3D resolution with maximum labelling efficiencies, absolute measurements of protein copy numbers, precise dual-colour measurements, high-throughput for large scale statistics and novel data analysis approaches, to address exciting biological questions, which were previously inaccessible.