Light microscopy is an extremely powerful tool for the life sciences. It allows extracting information about the architecture of biological systems in a very direct manner. Sample preparation and image acquisition are often simple. It is uniquely positioned to achieve molecular contrast, i.e. to highlight specific molecules in the sample. In multicolour measurements, the spatial relationships between cells, subcellular structures, and specific molecules can be visualized. Using fluorescent dyes or proteins as labels enables extremely high signal-to-background ratio with detection efficiencies that may reach the single fluorophore level.

Importantly, light microscopy is also suited to image living specimens, watching their dynamics and response to perturbations.

As a major drawback, conventional light microscopes are limited in their resolving power by the diffraction of light waves, such that features residing closer to each other than about half the wavelength of light or ~200 nm cannot be distinguished, thus limiting the analysis of fine structural details.

A series of powerful methods have been devised that enable optical resolution much better than the diffraction resolution limit. These “super-resolution” microscopy or “nanoscopy” approaches now routinely achieve resolution in the tens of nanometer range. They include single-molecule based methods like PALM and STORM and the coordinate-targeted methods like stimulated emission depletion (STED) microscopy. In STED microscopy, a pattern of light silences fluorophores except for those in immediate proximity of an intensity “zero” of the STED light pattern. It is overlapped with the excitation light pattern and thus defines regions of sub-diffraction extent where fluorescence is allowed to originate from. Further development of super-resolution imaging approaches with an emphasis on STED microscopy is a central focus of the group.

Analysis of entire cell or tissue volumes with diffraction-unlimited resolution in all three spatial directions is still a major challenge. Here, improved tissue imaging tools are warranted. Especially analysing living cells or tissues poses challenges for 3D super-resolution imaging. Biological validity will be further enhanced by approaches that mitigate light exposure and photobleaching and that improve 3D and repeated imaging capability. One such strategy is to protect fluorophores by using multiple off-state transitions for nanoscopy in an approach called “protected STED”. A link to the pertinent publication in Nature Photonics can be found here. Other challenges include visualizing multiple molecular players simultaneously to pinpoint their mutual relationships. We drive developments in these various directions inspired by a set of biological questions where these new tools promise to make a valuable contribution.

An interesting alternative approach to high-resolution imaging has recently emerged: rather than increasing instrument resolution, one can instead isotropically increase the physical separation between target molecules by embedding in and linking the sample to a swellable hydrogel that is then expanded. Such “expansion microscopy” enables effectively increased spatial resolution on conventional microscopes. We actively pursue developments in expansion microscopy. A detailed guide for optimization in X10 microscopy, published in Nature Protocols, can be found here. This variant of expansion microscopy enables a ten-fold resolution increase in each spatial direction in a single expansion step, thus increasing the sample volume 1000-fold.

In parallel to the instrumentation developments we work on novel labelling approaches to extract new types of information from the sample.

In terms of the biological applications, we put an emphasis on the analysis of tissues, on the interactions between cells, and the molecular make-up of their subcellular compartments. Especially for the analysis of neuronal tissue and the specialized signalling connections between neurons, i.e. the synapses, optical resolution beyond the diffraction limit is warranted.

For further questions and resources please contact Johann Danzl: