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 > Pharmaceutical Technology (Huwyler) |
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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 |
Identification Number: |
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Last Modified: | 21 Feb 2020 08:49 |
Deposited On: | 21 Feb 2020 08:49 |
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