The dynamic assembly of proteins must be spatially and temporally coordinated for essential processes to occur, from endo- and exocytosis, to cell division and organelle maintenance. Despite the importance of these processes, our physical paradigm for how proteins form mesoscale assemblies is far from complete. This is in part a consequence of technical limitations.
While the organization and dynamics of membrane proteins are heterogeneous, commonly used fluorescence-based measurements lack information at the molecular scale. In contrast, single molecule measurements limited to looking at only a few molecules in a given cell lack ensemble information. Thus, the study of protein assembly has been limited by a lack of spatially resolved, dynamic information on ensembles of molecules.
To overcome these obstacles, we develop and use automated super-resolution fluorescence imaging techniques combined with live cell imaging and single molecule tracking to determine how the dynamics of protein assembly are coordinated.
Large field-of-view super-resolution microscopy
Single molecule localization microscopies (SMLM) have been established as important tools for studying cellular features with resolutions on the order of ~10 nm. We have developed imaging modalities to perform large field-of-view SMLM using microlens array (MLA)-based flat-field epi-illumination. Our system can efficiently and homogeneously illuminate a large sample area (~150×150 µm2) enabling high-throughput SMLM imaging.
Multicolor 3D single particle reconstruction
Single-particle reconstruction from electron microscopy (EM) images is today able to reach atomic resolution. However, since the image contrast in EM is dependent upon the local electron density within biological structures, the resulting image lacks direct information on protein identity. We are developing computational and analytical frameworks to reconstructs and co-align multiple proteins from multicolor 2D SMLM images.
Waveguide TIRF for high-throughput DNA-PAINT
The PAINT method for localization microscopy offers higher localization precision and more continuous target sampling than standard photoswitching methods. We are using waveguides to increases throughput and data quality for PAINT. The waveguide chip is a multi-layer structure on a silicon substrate with dedicated sample wells to enable highly uniform ~100×2000 µm2 area evanescent field for TIRF illumination.
Mitochondria divide and fuse dynamically, and our aim is to unravel the physical and physiological signatures of these processes. By quantifying the dynamic geometry of mitochondrial constrictions, we extract the energies and forces required for division and link them to interactions with the division machinery. Thereby, we can develop a physical framework accounting for the energetic requirements for fission. Furthermore, we are investigating the metabolic and bioenergetic changes associated with mitochondrial division on a single-organelle level. A custom built instant-SIM enables us to perform fast, multicolor imaging on whole cells.
Organization of mitochondrial gene expression
Each mitochondria has its own genome (mtDNA) that needs to be transcribed and translated in a regulated manner. Interestingly, mitochondrial RNA accumulates in foci termed Mitochondrial RNA Granules (MRGs). However, little is known about the biophysical properties of MRGs and their associated proteins. We aim to elucidate the structural, dynamic, and regulatory properties of MRGs to better understand their role in mitochondrial physiology.