Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins
With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nan...
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| Hauptverfasser: | , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
2018
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| In: |
Scientific reports
Year: 2018, Jahrgang: 8 |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/s41598-018-23818-0 |
| Online-Zugang: | Verlag, kostenfrei, Volltext: http://dx.doi.org/10.1038/s41598-018-23818-0 Verlag, kostenfrei, Volltext: https://www.nature.com/articles/s41598-018-23818-0 
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| Verfasserangaben: | Varun Venkataramani, Markus Kardorff, Frank Herrmannsdörfer, Ralph Wieneke, Alina Klein, Robert Tampé, Mike Heilemann & Thomas Kuner |
| Zusammenfassung: | With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the ‘labeling barrier’ and to bypass photobleaching in multi-plane, whole-cell 3D experiments. |
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| Beschreibung: | Published online: 03 April 2018 Gesehen am 14.06.2018 |
| Beschreibung: | Online Resource |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/s41598-018-23818-0 |