What is the problem/issue being addressed?
Fluorescence microscopy is a powerful tool for visualizing biomolecules in cells and tissues, especially with the advent of super-resolution techniques (e.g. STED, PALM, or STORM) enabling spatial resolutions of 10–100 nm. These techniques have already had remarkable success in revealing hidden details of subcellular organelles and structures and are poised to have a profound impact in biological and biomedical research moving forward.
Investigating the localization, abundance, and mutual interactions of central cellular components such as nucleic acids and proteins are crucial for cell differentiation, disease progression and the fate of cells. Additionally, in multicellular organisms, extensive changes in the proteomic signature are governed by cell-cell communications in tissue microenvironments. To understand complex cellular processes in situ, it is therefore not only crucial to obtain molecular identities from a multitude of key proteins and nucleic acids, but equally important to spatially localize them relative to other components in- and outside the cell.
However, in fluorescence and especially super-resolution microscopy the number of distinct molecular species that can be measured simultaneously (i.e. the multiplexing power) was typically limited by the spectral overlap between fluorophores to 3–4 species. Furthermore, although breaking the classical diffraction limit of light, incumbent super-resolution techniques were only able to resolve 20-nm-distances under ideal conditions. Finally, an important, unsolved issue evolved around the labeling probes in high-resolution microscopy applications, quickly becoming the bottleneck in translating technical capabilities to cellular applications. We pioneered the development of DNA-PAINT super-resolution microscopy, which uses the molecular recognition capability and programmability of DNA molecules. In DNA-PAINT, stochastic switching between fluorescence on- and off-states is facilitated by repetitive, transient binding of short fluorescently labeled oligonucleotides (“imager” strands) to complementary “docking” strands.
What are the overall objectives?
To overcome the limitations of current super-resolution implementations, I proposed to advance DNA-based microscopy in key areas to enable highly multiplexed, high-resolution imaging for a multitude of cellular components, thus paving the way for applications of super-resolution in biomedically relevant research areas.
Importance to society?
The development of novel DNA-based imaging capabilities will provide the tools to answer open questions in the life sciences, which were thus far elusive. Thus, one clear societal benefit is the creation of new textbook knowledge used to educate the next generation of life science researchers. Furthermore, the tools developed in this ERC Starting Grant have been made publicly available (both in terms of reagents and assays as well as analysis software) following best open science practice, such that the scientific research community gained direct access to state-of-the-art developments from this ERC grant. Finally, techniques developed in this project could become a door opener to eventually allow almost proteome-wide classification of disease states, help devise therapeutic decisions as well as monitor therapeutic responses.
Conclusions
In conclusion, the research carried out in this ERC Grant has help tremendously to push DNA-based super-resolution microscopy to the next level, paving the way to quantitative high-throughput studies under biomedically relevant conditions. While we were able to advance spatial resolution to approx. 5 nm as well as improve the labeling probes and image acquisition speed, there is still a dire need in carefully benchmarked, improved labeling probes for next-generation super-resolution microscopy, an area where we actively develop new approaches at the moment.
