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 e.g. by the size of organelles or multi-molecular complexes. Single-molecule localisation-based superresolution 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 superresolution 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. We used this approach to study DNA structures and alpha-synuclein amyloids and could demonstrate 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 superresolution microscopy. This will allow us to extract information about protein structures from superresolution microscopy data.
Future projects and goals
Our vision is to establish superresolution 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. To this end, we have to push its limits on all fronts to establish a technique which combines nanometer three-dimensional resolution with maximum labeling efficiencies, absolute measurements of protein copy numbers, precise dual-color measurements, high-throughput for large scale statistics and novel data analysis approaches. With these tools we can address exciting biological questions, which were previously inaccessible.