Optical nanoscopy

Development of super-resolution optical microscopy techniques

Despite the superior spatial resolving capabilities of electron microscopy, vast majority of all microscopy applications in life sciences is performed using light microscopy. However, the resolution of a standard far-field light microscope, as first realised by Ernst Abbe in the late 19^th century (Abbe 1873), is limited to about one third of the wavelength, because smaller focal spots could not be formed with high numerical aperture objectives due to diffraction. For visible light, this means approximately 180nm minimum distance between two objects. Shorter wavelengths can yield higher resolution, but live cells often suffer from irradiation caused by light at ultra-violet and shorter wavelengths.

Recent research pioneered in the mid 1990's by the Laboratory of Biophysics, in form of a so called STED microscope (Hell & Wichmann 1994), has proven that the fundamental diffraction barrier can be surpassed in fluorescence far-field microscopy, making close to molecular-scale resolutions available.s.

Our research effort aims to further develop different super-resolution imaging technologies. RESOLFT technology (Hell 2007) based on the basic principle of Stimulated Emission Depletion Microscopy (STED) will be further developed, taking advantage of multi-photon processes in the activation and de-activation of fluorescent molecules. Alongside the RESOLFT development effort, a state-of-the-art ?optical toolbox" will be developed to include different statistical and photo-activation based high-resolution localisation techniques, combined with total internal reflection fluorescence (TIRF) microscopy.

Analysis of bone remodeling process by utilising modern high-resolution microscopy techniques

Modern, non-destructive imaging- and imaging related techniques such as the Atomic Force Microscopy (AFM), Confocal Fluorescence Microscopy (CFM), Total Internal Reflection Fluorescence Microscopy (TIRF), Multiphoton Fluorescence Microscopy (MFM), Stimulated Emission Depletion Microsopy (STED) and Multimode Fluorescence Detection (MFD) enable dynamic studies of (live) biological phenomena down to single molecule scale. These techniques have largely been applied individually on particular problems. However since ?real world" problems often require information from multiple, independent sources, multimodal approaches in the use of these techniques have started to emerge. The project aims to make use of a combination of exciting recent advances in the field of microscopy for visualization and analysis of cell membrane and structure associated with bone remodeling process.

Bone is a dynamic and fascinating tissue that undergoes continuous remodelling throughout life. Bone-resorbing osteoclasts are highly polarized that resorb bone by generating a pH gradient between the cell and the bone surface. During bone remodelling, bone resorption and bone formation are tightly coupled via as yet uncharacterised factors. The purpose of this research program is to develop multimodal instrumentation and methods for the study of osteoclast resorption (binding) and bone formation phenomena. The projects consists of three parts: visualization and characterization of the osteoclastic bone binding domains and their function down to single molecule level, development of new tracer molecules by recombinant antibody library techniques and study of the biological function of the binding domain (sealing zone). The project involving local experts in biological multimodal imaging, recombinant library techniques and bone biology is carried out in collaboration with international leading groups in force microscopy, single molecule detection and high-resolution light microscopy.

Since osteoclast binding is a highly specific and a unique function of these cells, better understanding of the function may open up new diagnostic techniques for early detection of dysfunction in the remodeling process (osteoporosis) and even suggest new highly specific drug targets. The instrumental methods and results of the project may also be exploited in study of other complex biological phenomena from sub-cellular to single molecule level.

Nanoscopy research group

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