Figure 1: Localisation microscopy of proteins from a GFP-fusion construct library in budding yeast. Nic96: component of nuclear pore complex (NPC), individual NPCs are visible as ringlike structures. Spc42: part of the spindle pole body. Diffraction limited images (green) and reconstructed superresolution images (red) are shown.
Figure 2: Dual-color superresolution images of Cdc11 (red) and the cell-wall marker ConA (green) show the formation and disassembly of the Cdc11 ring
Click on image for larger version.
The Ries group develops cutting-edge superresolution microscopy methods, such as automated localisation microscopy for proteome-wide superresolution imaging.
Previous and current research
The resolution of optical microscopy is limited by diffraction to about 200 nm, which is much larger than the relevant length-scales in cell biology, defined for instance by the size of organelles or supramolecular complexes. Single molecule localisation-based superresolution microscopy methods, such as photoactivated localisation microscopy (PALM), rely on the stochastic activation and subsequent localisation of individual fluorophores. They reach a 10-fold higher resolution, which is optimal for the study of intracellular structures. Until now, however, these techniques required special fluorescent proteins to be cloned or high-affinity antibodies to be generated for specific labelling. On the other hand, many laboratories have most of their constructs in green fluorescent protein (GFP) form and entire genomes are available as functional GFP-fusion proteins. We recently developed a labelling scheme to make all these constructs immediately available for superresolution microscopy by targeting them with small antibodies labelled with bright organic dyes. This opens the door to high-throughput localisation analysis of entire genomes at the nanoscopic level in cells.
Our current research efforts are threefold. First, collaborating closely with other groups, we are establishing state-of-the-art superresolution microscopy to answer exciting questions in cell biology, which have only now become accessible due to greatly improved resolution. Second, we are working on automating single-molecule localisation microscopy with the aim of proteome-wide imaging – such superresolution localisation maps of proteins will be an invaluable resource for the life science community. Third, we are developing novel detection schemes for localisation microscopy. In one project we intend to use the principle of surface-generated fluorescence to improve the axial resolution. In another we aim to measure the 3D orientation of single molecules, in addition to their position by polarised detection, in order to resolve the structure of multi-molecular complexes.
Future projects and goals
Our goal is to establish cutting-edge superresolution microscopy and to apply it to biological systems. We will implement the newly developed detection schemes in a powerful microscope and combine this with advanced data analysis and our expertise in sample preparation and labelling. Using automated single-molecule localisation microscopy we are planning to image the whole proteome of budding yeast with a resolution of ~20 nm in dual-color and 3D. The combination of optical superresolution microscopy with dynamic microscopy techniques such as fluorescence correlation spectroscopy (FCS) or single particle tracking (SPT) bears great potential in relating structure, localisation and function, as does the combination with electron microscopy to add molecular specificity to the ultra structure.