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From Tissues to Single Molecules: High Content in Situ Super-Resolution imaging with DNA-PAINT

Periodic Reporting for period 3 - MolMap (From Tissues to Single Molecules: High Content in Situ Super-Resolution imaging with DNA-PAINT)

Reporting period: 2019-04-01 to 2020-09-30

Fluorescence microscopy is a powerful tool for exploring biomolecules in cells and tissues, especially with the advent of super-resolution techniques. To better understand key processes such as cell differentiation and disease progression, it is crucial to investigate the abundance, localization and mutual interactions of crucial cellular components such as nucleic acids and proteins. Unraveling their complex interplay in whole signaling networks is necessary to investigate cellular responses to stimuli.

However, currently available characterization techniques are either limited by low multiplexing capability (e.g. fluorescence microscopy) or lack localization information (e.g. mass spectrometry). Despite the immense biological and clinical relevance of understanding network-wide changes, the lack of a technological platform to image, identify and quantify a multitude of key protein networks at high spatial resolution in tissues impedes our understanding of the molecular basis of health and disease.

The overall goal of this project is to solve this pressing issue and revolutionize fluorescence microscopy using tools from DNA Nanotechnology with transformative potential to positively answer the question: Can we localize and identify each protein or nucleic acid molecule in a complex tissue microenvironment?
The approach is based on recently developed DNA-based imaging techniques. To push the envelope of what’s technically possible, we will advance state-of-the-art instrumentation for deep tissue high throughput DNA-PAINT imaging. We will furthermore develop novel affinity-based labeling approaches in combination with molecular barcoding and automated multiplexed image acquisition and processing in order to reach our goal of imaging whole biomolecular networks in cells and beyond with a fluorescence microscope.
With these disruptive and transformative tools, we will ultimately investigate whole signaling cascades at once in single cells and whole tissues, thus enabling quantitative imaging with highest spatial resolution.
Towards our final goal of developing a platform for imaging proteomics and transcriptomics, we have thus far achieved several key milestones.
We have made solid progress towards the implementation of DNA-based super-resolution microscopy using optical sectioning microscopy such as advanced Light Sheet and Spinning Disk Confocal, while the letter implementation was recently published. This now allows us to observe cellular systems throughout the thickness of whole cells and beyond with exquisite spatial resolution and spectrally-unlimited multiplexing, thus far impossible to achieve.
In order to obtain high-throughput imaging capabilities that will allow us to screen for rare events in tissue biology while maintaining high multiplexing capabilities, we have implemented our DNA Exchange approach – published for DNA-PAINT super-resolution microscopy a few years ago – for a large variety of fast imaging approaches such as standard confocal, widefield and commonly-used super-resolution approaches.
Towards the goal of quantitative and highly multiplexed super-resolution microscopy, we have achieved several milestones, including quantitative counting of biomolecules in vitro and in situ. We now have the technical prerequisites to put integer numbers on protein clusters throughout the cell, opening the door to single-molecule Systems Biology research in single cells and beyond.
A major cornerstone of the proposed ERC work was the development of novel, small, and efficient binders for labeling proteins with DNA molecules with highest specificity, affinity, and accuracy. To this end, we have developed and assayed novel DNA-conjugated protein binders such as primary and secondary antibodies, small protein binders called Affimers, and novel DNA-based protein binders called aptamers. Together, these biochemical labeling reagents allow us and for the matter of fact many researchers worldwide, to ask cell biological and biomedical questions that were thus far elusive. One no longer has to limit oneself to the interrogation of just a handful of cellular targets. Now, we can ask the question about how whole networks and molecular systems of proteins and nucleic acids interact and influence each other at highest spatial resolution.
The research performed thus far within the realm of this ERC Starting Grant lays the technological groundwork for the proposed application of DNA-based fluorescence microscopy to image hundreds to thousands molecular species in single cells and tissues. It has clearly gone beyond state-of-the-art in the way we look at fluorescence imaging in general and single-molecule super-resolution microscopy in particular. Through our advancements in molecular probe design, we were able to transform traditional multicolor fluorescence microscopy into a sequence-programmable DNA-based imaging system that allows researchers to image tens to hundreds of molecular targets in single cells using just a single-color dye and laser system.
We have furthermore tackled one of the major unsolved problems of high-resolution fluorescence microscopy, which is the availability of small, efficient, and quantitative labeling reagents for targeting protein molecules in cells. We have combined unique affinity binding molecules called aptamers – DNA analogs of antibodies – with our unique DNA-based super-resolution technique to potentially provide the ultimate quantitative labeling probe for fluorescence super-resolution microscopy.
We will now further advance instrumentation and molecular barcoding to reach our ultimate goal of obtaining a complete atlas of single cells and tissues at highest spatial resolution and multiplexing. This will involve advancements and application of Lattice Light Sheet microscopy, automated fluid exchange, kinetically-encoded simultaneous barcoding and testing and verification of a large library of thousands of aptamer reagents for super-resolution imaging.