Combining advanced fluorescence methods and nanoagents

In this project, we combine advanced fluorescence methods and nanoagents. On the one hand, single molecule methods make it possible to observe the functionality of individual nanoagents, making it possible to improve the nanoagents. On the other hand, nanoagents can be used to provide complementary capabilities or enhance different fluorescence methods. During the second funding period, we have applied different advanced fluorescence to investigate the function of novel nanoagents and also developed nanoagents to provide new functionalities for single-molecule experiments. The junior group leader, Dr. Ploetz, has built a Raman microscope, which we can use to investigate nanoagents that cannot be fluorescently labeled. The success of the last funding period has so far resulted in 16 SFB1032 related publications with five groups within the SFB and results from collaborations with another two groups are currently being prepared for publication. During the current funding period, we will build upon the successes of the last funding period. We will continue to use nanoagents to improve fluorescence methodologies and use our expertise in advanced fluorescence methods to visualize and better understand how they function.

The first group of projects focuses on the application of nucleotides as nanoagents. We will extend the work with Friedrich Simmel (A02) on the roboarm for nano assembly lines. Using 3D orbital tracking, we will follow the motion of the roboarm in real-time in 3D and investigate how freely the arm can rotate and whether there are non-desired interactions with the DNA platform. We will also follow transfer of cargo from one roboarm to the next using two- and three-color FRET. In collaboration with Thomas Carell (A05), we will use multi-photon microscopy to follow the uptake and distribution of cyclic nucleotides in living cells, which can be used to regulate signaling pathways in cells. Metal enhanced fluorescence can boost the fluorescence signal available from a single fluorophore. Together with the group of Philip Tinnefeld (A13), we will use DNA-origami to position a protein in the hotspot between two metal nano-particles. Thereby, we can enhance the radiate rate of a particle and thus follow the dynamics of proteins directly on the sub-millisecond timescale using FRET within the hotspot.
In the next projects, we will use light to control biological properties and cellular processes. Continuing our collaboration with Theobald Lohmüller (A08), we will investigate how the properties of lipid bilayers change on the single-molecule scale with his photoswitchable lipids. We will also combine the photoswitchable lipids with super-resolution techniques to generate sub-diffraction sized artificial lipid rafts. These will be great test systems for investigating the presence and effect of small lipid rafts in membranes. In a second project, we will use the LOV constructs (van Bergeijk et al. Nature. 2015, 518: 111–114.) or photoswitchable constructs provided from Oliver Thorn-Seshold (B09) to control the binding of motors to cargo. Using our 3D orbital tracking setup, we will then follow the changes in the transport behavior depending upon the presence or absence of particular motor proteins. Thereby we hope to gain insights into how motor proteins communicate.
A third direction we will pursue involves label-free imaging approaches for single-particle tracking, in which we complement our fluorescence methods with nonlinear Raman imaging. The combined fluorescence/Raman microscope provides simultaneous chemical sensitivity and single-molecule precision in living cells. With this system, we will follow the uptake and degradation of metal-organic framework based nanoparticles in cells by tracking. Raman scattering will be used to extract chemical information regarding the composition of the nanoparticles as a function of time within the cellular context. In another project, we will combine the expertise of Dr. Ploetz with respect to Raman and our orbital tracking methodology to be able to track nanoparticles indefinitely without photobleaching.