Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins

• Verfasst von: Venkataramani, Varun [VerfasserIn]
• Verfasst von: Kardorff, Markus [VerfasserIn]
• Verfasst von: Herrmannsdörfer, Frank [VerfasserIn]
• Verfasst von: Heilemann, Mike [VerfasserIn]
• Verfasst von: Kuner, Thomas [VerfasserIn]
• Titel: Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins
• Verf.angabe: Varun Venkataramani, Markus Kardorff, Frank Herrmannsdörfer, Ralph Wieneke, Alina Klein, Robert Tampé, Mike Heilemann & Thomas Kuner
• Jahr: 2018
• Umfang: 7 S.
• Illustrationen: Illustrationen
• Fussnoten: Published online: 03 April 2018 ; Gesehen am 14.06.2018
• Titel Quelle: Enthalten in: Scientific reports
• Ort Quelle: [London] : Macmillan Publishers Limited, part of Springer Nature, 2011
• Jahr Quelle: 2018
• Band/Heft Quelle: 8(2018) Artikel-Nummer 5507, 7 Seiten
• ISSN Quelle: 2045-2322
• DOI: doi:10.1038/s41598-018-23818-0
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1576373878
• K10plus-PPN:
  Universitätsbibliothek
• Lokale URL UB: Zum Volltext

Abstract

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.