Nucleic-Acid Based Super-Resolution Microscopy

• Applicant: Professor Dr. Ralf Jungmann
• Subject Area: Biophysics
• Term: from 2014 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 251122678

In his 1959 talk There is plenty of room at the bottom, Richard Feynman posited that with a sufficiently powerful microscope ...it would be very easy to answer many fundamental biological questions; you just look at the thing!Many processes in biological systems involve the interaction of single molecules on the nanometer scale, and thus, the ultimate goal of microscopy is the observation of these processes with high resolution. While it is possible to detect a signal from a single biomolecule with a fluorescent tag, conventional far-field fluorescence techniques are limited by the diffraction limit to a resolution of about 200 nm. However, all of this changed in the 1990s with the advent of super-resolution techniques allowing for the circumvention of the diffraction limit. Although current techniques hold promise to broadly transform biomedical research and allow imaging with up to 10-times higher spatial resolution, they still fall short in four important areas:To start, existing techniques lack true biomolecular resolution, preventing the observation of interactions of densely organized single molecules as found for example in surface receptor clusters. Secondly, traditional imaging approaches offer only low multiplexing power, i.e. the ability to observe a multitude of molecules simultaneously. Thirdly, while the real power of fluorescence imaging lies in its potential to visualize processes in living cells, live cell super-resolution imaging remains limited in scope and challenging to implement (e.g. delivering synthetic imaging probes to living cells in a biocompatible fashion is a common hurdle). Finally, current methods tend to require either expensive instrumentation or specialized experimental conditions, limiting their application in a standard biological lab environment.My proposed research program is aimed at advancing current super-resolution techniques to specifically address these shortcomings. I will do this by integrating single-molecule fluorescence techniques with the unique programmability and specificity properties of DNA molecules, leading to a new kind of molecular fluorescent probe based on engineered properties. The novel nucleic acid-based probes will allow imaging on a molecular scale (<5 nm) while offering unprecedented multiplexing power via a novel approach based on blinking frequency encoding, a common concept in biology (e.g. fireflies talk via blinking). I will implement this concept using autonomously blinking DNA-based probes for the purpose of barcoding molecules. I will also develop a genetically encoded live-cell compatible super-resolution technique based on genetically encoded RNA sequences that transiently bind a non-cytotoxic small conditional dye.

• DFG Programme: Independent Junior Research Groups
• Major Instrumentation: automated fluorescence microscope ; module for single-molecule super-resolution
• Instrumentation Group: 5040 Spezielle Mikroskope (außer 500-503) ; 5080 Optisches Mikroskopzubehör