Final Report Summary - BRAIN STED (Intravital optical super-resolution imaging in the brain) Our understanding of the fundamental processes of life and the mechanisms of disease is progressing at a rapid pace. This dynamic process is largely fuelled by the availability of novel technologies for analysing and manipulating biological matter. In this context, optical microscopy exerts an important function because it allows the direct visualization of the dynamics of life in cells, tissues, and even organisms. In particular, fluorescent tags can be employed for labelling of biological structures and hence conferring molecular specificity to the observation. For the analysis of living biological systems, genetically encoded markers such as green fluorescent protein have proven extremely powerful. However, the spatial resolution of optical microscopes, i.e. their ability to discern nearby features, is fundamentally limited by optical diffraction to about half the wavelength of light and therefore not adequate for addressing many biologically relevant questions. Accordingly, in the biomedical field, there is a soaring need for powerful optical techniques with diffraction-unlimited spatial resolution that allow the dissection of the nanoscale organization of biological matter. Several far-field super-resolution imaging techniques have been developed. Among these, coordinate-targeted methods, including stimulated-emission-depletion (STED) microscopy and the generalized RESOLFT concept, stand out by their ability to capture fast dynamics and the possibility to image inside living tissue. Coordinate-stochastic methods such as STORM, PALM, and GSDIM also provide high spatial resolution but are less suited to capture dynamics and have so far not been applicable to tissue specimens. We set out to develop novel strategies that would allow for fluorescence imaging of nanoscale structure and dynamics deep inside tissue specimens, most prominently in the brain. We first evaluated a series of existing technologies for nanoscopy with genetically encoded markers with the aid of a dedicated home-built microscope. Based on this analysis, we realized that it might be possible to achieve superior imaging performance with a novel super-resolution imaging approach and developed a strategy that might enable improved resolution, signal-to-noise ratio, and long-term imaging capability paired with the ability to image in living tissue samples. Encouraged by initial tests of this approach, we constructed a super-resolution microscope based on this concept and tested it with technical samples and with living cell culture specimens. As an important result, we were able to demonstrate that the method does indeed fulfil the criteria of competitive resolution, high image quality, and the capability of repeated imaging. We next tested whether the method would also be suitable for imaging neuronal cells in cultured brain tissue. To this end, the optical aberrations introduced by the nervous tissue had to be compensated for. We were able not only to image the most superficial cell layers but also to achieve high-quality imaging of neurons deep within cultured tissue. In conclusion, we have developed a novel nanoscopy approach that we expect to outperform existing methods in several key aspects. Importantly, the method specifically aims at imaging living, dynamical biological systems, rather than having to rely on fixed samples. The motivation for improving nanoscopy technology was clearly driven by the potential benefit in neuroscience. We hope that the new method will allow imaging of synaptic structure in unprecedented detail in living tissue and that it will be able to capture the subtle but important dynamical changes associated with brain activity. Similarly, as soon as a two-color implementation will be put into operation and hence the distribution of two molecular entities can be discerned, it should be possible to image the intricate interaction between different neurons or between neurons and glia both in the healthy and in the diseased brain. However, we expect that the impact of the method will not be limited to neuroscience but will be more general. We anticipate that it will be applicable to a wide range of biomedical problems, wherever it is necessary to decode the nanoscale details of the structure of a living specimen and its dynamical time evolution, be it in isolated cells, in tissue samples or in whole living model organisms. There is an increasing need for superior high-resolution fluorescence imaging modalities in the biomedical field. Apart from its impact in basic research, the method might add to European competitiveness in the growing market of super-resolution microscopy for biomedical applications since it will likely be of interest to microscope manufacturers. It is well conceivable that the method will, after an additional development period carried out at academic institutions or in partnership with industry, be commercially distributed and prove competitive in an important segment of that market.