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Multicolor super-resolution imaging with 3D structured illumination microscopy

Dr. Lothar Schermelleh
LMU Munich

 

Studies of subcellular structures using state-of-the-art light microscopy have been restricted by the diffraction barrier of optical resolution that is ~250 nm in the xy‑plane and ~600 nm along the z-axis. Recent developments such as stimulated emission depletion (STED) microscopy and stochastic localization microscopy (STORM/PALM) have made possible to surpass Abbe’s diffraction limit. An alternative approach developed by John Sedat and colleagues at the UCSF uses three-dimensional structured illumination (3D-SIM) to improve lateral as well as axial resolution by a factor of two below the diffraction limit. This technology has been implemented in a specially designed microscope platform, termed OMX, which provides unprecedented sensitivity and mechanical stability. To explore the potential of the 3D-SIM technology we have applied the OMX prototype on a wide variety of biological structures. I will present the conceptual basics of the OMX microscope and present high-resolution data on various structural features in mammalian cell nuclei, including multi-fluorescence 3D data on the ultra-structural organization of the nuclear envelope and chromatin. The results clearly demonstrate the potential of the OMX microscope for multi-wavelength optical sectioning of biological samples with subdiffraction resolution that will allow new insights in biological structures and will help to narrow the gap between light and electron microscopy.