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

Venkataramani, Varun and Kardorff, Markus and Herrmannsdörfer, Frank and Wieneke, Ralph and Klein, Alina and Tampé, Robert and Heilemann, Mike and Kuner, Thomas. (2018) Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins. Scientific Reports, 8 (1). p. 5507.

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Official URL: https://edoc.unibas.ch/75630/

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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.
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Pharmazeutische Technologie (Huwyler)
UniBasel Contributors:Kardorff, Markus
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:Nature Publishing Group
ISSN:2045-2322
e-ISSN:2045-2322
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:21 Feb 2020 08:49
Deposited On:21 Feb 2020 08:49

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