Struktur und Dynamik des Kernporenkomplex in lebenden Zellen
Final Report Abstract
The crux of our findings is to combine the advantages of light microscopy (LM), namely the imaging of cellular processes in real time and the capability to unambiguously designate the underlying protein by fluorescence markers, with the benefit of electron microscopy (EM), namely visualizing the ultrastructural context in high resolution. The protocol, we developed and established both for laboratory ex vivo cell cultures and for various tissues allows visualizing both individual subcellular structures and the overall tissue context at high quality without the need to compromise on the optimal EM preparation conditions. The protocol also allows the use of latest generation super-resolution LM techniques, which are no longer restricted in the differentiation between two image points by the wavelength of the light. At the same time, both the imaging at EM resolution for bigger fields of view by advanced scanning EM and for high resolution 3D EM by transmission EM tomography is possible. Furthermore, we showed both extracellular and intracellular (by using cell membrane permeable dyes) fluorescent multicolor labeling of structures. Electron microscopy provides the link between LM imaging and techniques allowing visualizing macromolecules at atomic resolution. Our techniques allow merging these techniques without needing to compromise on the quality of the EM preparation. Hence, given the recent technological advances in the EM field (convenient EM preparation systems, improved quality microscopes and detectors) and the growing demand of natural sciences to link physiological and biochemical findings to the underlying cellular structure, we expect that our protocol will find widespread utilization. We early on followed the protocols based on fluorescent proteins used by others in the field. However, while evaluating our results, we found both the level of fluorescence that survived the EM preparation protocols and the need to compromise on ultrastructural preservation to preserve fluorescence not acceptable. Hence, we early on switched to the chemical fluorophores. The high level of fluorescence preservation of the chemical fluorophores in the final EM samples and the fact that also super-resolution LM proofed to be still possible, was for us a not foreseeable but important finding.
Publications
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(2014): Correlative light- and electron microscopy with chemical tags. In: J. Struct. Biol. 186 (2), S. 205–213
Perkovic, Mario; Kunz, Michael; Endesfelder, Ulrike; Bunse, Stefanie; Wigge, Christoph; Yu, Zhou; Hodirnau, Victor-Valentin; Scheffer, Margot P.; Seybert, Anja; Malkusch, Sebastian; Schuman, Erin M.; Heilemann, Mike; Frangakis Achilleas S.
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(2014): Super-sampling SART with ordered subsets. In: Journal of structural biology
Kunz, Michael; Frangakis, Achilleas S.
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PLEKHM1 regulates Salmonella-containing vacuole biogenesis and infection. In: Cell Host & Microbe, Volume 17, Issue 1, 14 January 2015, Pages 58-71
McEwan, David G.; Richter, Benjamin; Claudi, Beatrice; Wigge, Christoph; Wild, Philipp; Farhan, Hesso; McGourty, Kieran ; Coxon, Fraser P.; Franz-Wachtel, Mirita; Perdu, Bram; Akutsu, Masato; Habermann, Anja;Kirchof, Anja; Helfrich, Miep H.; Odgren, Paul
