RESOLFT Nanoscopy of Fixed Cells Using a Z-Domain Based Fusion Protein for Labelling |
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Authors: | Peter Ilgen Tim Grotjohann Daniel C Jans Markus Kilisch Stefan W Hell Stefan Jakobs |
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Institution: | 1. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.; 2. Department of Neurology, University Medical Center of Göttingen, Göttingen, Germany.; 3. Department of Molecular Biology, University Medical Center of Göttingen, Göttingen, Germany.; University of Manchester, UNITED KINGDOM, |
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Abstract: | RESOLFT super-resolution microscopy allows subdiffraction resolution imaging of living cells using low intensities of light. It relies on the light-driven switching of reversible switchable fluorescent proteins (RSFPs). So far, RESOLFT imaging was restricted to living cells, because chemical fixation typically affects the switching characteristics of RSFPs. In this study we created a fusion construct (FLASR) consisting of the RSFP rsEGFP2 and the divalent form of the antibody binding Z domain from protein A. FLASR can be used analogous to secondary antibodies in conventional immunochemistry, facilitating simple and robust sample preparation. We demonstrate RESOLFT super-resolution microscopy on chemically fixed mammalian cells. The approach may be extended to other super-resolution approaches requiring fluorescent proteins in an aqueous environment. |
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