Periodic Reporting for period 4 - MolMap (From Tissues to Single Molecules: High Content in Situ Super-Resolution imaging with DNA-PAINT)
Reporting period: 2020-10-01 to 2022-03-31
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.
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.
During the ERC Starting Grant project, my group was able to advance DNA-based super-resolution microscopy in key areas, overcoming limitations of incumbent super-resolution approaches and paving the way for highly multiplexed super-resolution imaging with applications in the life sciences.
Specifically, the main research and technological achievements of our approach resulted in
1) Multiplexed DNA-based super-resolution microscopy in whole cells, away from the coverslip interface, paving the way for efficient tissue-scale super-resolution imaging (Nature Communications, 2017)
2) Fast, spectrally unlimited multiplexed imaging for standard diffraction-limited fluorescence microscopy as well as STED, STORM, and SIM microscopy (Angewandte Chemie, 2017)
3) Highly-multiplexed, simultaneous target detection using engineered blinking frequency barcodes, enabling – for the first time – 124-plex super-resolution imaging (Nano Letters, 2019)
4) Development of genetically encoded as well as novel aptamer-based labeling probes for efficient and specific labeling of protein targets (Angewandte Chemie, 2019; Nature Methods, 2018)
5) Applications of the newly developed probes and imaging capabilities to unravel a novel function of the nucleolus in protein quality control (Science, 2019)
6) Considerable speed improvement by de novo sequence design yielding a 10-fold faster image acquisition speed (Nature Methods, 2019)
7) Finally, we were able to provide a comprehensive suite of labeling and imaging protocols alongside an open-source analysis software called Picasso to allow the scientific community to quickly adapt our improved imaging capabilities (Nature Protocols, 2017)
Specifically, the main research and technological achievements of our approach resulted in
1) Multiplexed DNA-based super-resolution microscopy in whole cells, away from the coverslip interface, paving the way for efficient tissue-scale super-resolution imaging (Nature Communications, 2017)
2) Fast, spectrally unlimited multiplexed imaging for standard diffraction-limited fluorescence microscopy as well as STED, STORM, and SIM microscopy (Angewandte Chemie, 2017)
3) Highly-multiplexed, simultaneous target detection using engineered blinking frequency barcodes, enabling – for the first time – 124-plex super-resolution imaging (Nano Letters, 2019)
4) Development of genetically encoded as well as novel aptamer-based labeling probes for efficient and specific labeling of protein targets (Angewandte Chemie, 2019; Nature Methods, 2018)
5) Applications of the newly developed probes and imaging capabilities to unravel a novel function of the nucleolus in protein quality control (Science, 2019)
6) Considerable speed improvement by de novo sequence design yielding a 10-fold faster image acquisition speed (Nature Methods, 2019)
7) Finally, we were able to provide a comprehensive suite of labeling and imaging protocols alongside an open-source analysis software called Picasso to allow the scientific community to quickly adapt our improved imaging capabilities (Nature Protocols, 2017)
All objectives and the resulting publications have gone far beyond the state of the art in super-resolution microscopy as present at the beginning of the project. Before our grant, super-resolution multiplexing was limited to a handful of targets at or above 20 nm spatial resolution close to the coverslip interface. We have pushed the envelope on all fronts enabling spectrally unlimited multiplexing at sub-10-nm resolution throughout whole cells and applied the newly developed techniques to cell-biological questions.