Conventional fluorescence microscopy is one of the major instrumentation offering a high spatiotemporal resolution and high molecular contrast necessary to elucidate cell functions. However, the optical resolution is far not sufficient for visualization and investigation of subcellular structures such as organelles (0.1-1um) or even individual proteins (1-10nm). Super-resolution microscopy enables to obtain high-resolution images beyond the diffraction limit. Broader use of super-resolution fluorescence microscopy promises an observation of previously unresolved details, but its applicability to various biological tasks still remains challenging. In a collaboration with biomedical imaging laboratory of Prof. Lasser (LOB, EPFL, Switzerland) and laboratory of Dr. Hagen (Institute of Cellular Biology and Pathology, Charles University, Czech Republic), we are focused on optimization and further development of structured illumination microscopy techniques (SIM) and super-resolution optical fluctuation imaging towards fast 3D live cell imaging.
In cooperation with the Prof. Lasser’s team, we are going to extend the imaging method bSOFI, which was introduced by Dr. Geissbuehler under the leadership of Prof. Lasser. bSOFI has a great potential to achieve high quality images even for weak signals and high labeling densities. This is very important for imaging live, complex cellular structures labeled by fluorescent proteins.
A more detailed examination of the influence of sensing and signal parameters on the imaging performance as well as improved image processing will allow broader applicability of the bSOFI method. We intend to perform 3D super-resolution imaging of live mitochondrial networks in a unique spatio-temporal resolution. High-resolution imaging of mitochondrial networks in different physiological conditions appears as a great challenge and opportunity opening new avenues in the understanding of age-related pathologies such as Parkinson’s disease and type 2 diabetes which are linked to major changes in mitochondrial function.
Miloš Klíma, Petr Páta, Karel Fliegel, Tomáš Lukeš, Petr Janout, Jan Bednář
EPFL – Ecole Polytechnique Fédérale de Lausanne, Lausanne (Switzerland)
Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague (Czech Republic)
Lukeš, T.: Sciex Fellowship: MitoSOFI – Super-resolution Optical Fluctuation Imaging of Mitochondrial Networks (Project Code 13.183) (2014 – 2015)
Lukeš, T.: Super-resolution microscopy imaging of mitochondrial networks 14–15, SGS SGS14/148/OHK3/2T/13 (2014 – 2015)