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 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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Author:Varun Venkataramani, Markus Kardorf, Frank Herrmannsdörfer, Ralph WienekeORCiDGND, Alina Klein, Robert TampéORCiDGND, Mike HeilemannORCiDGND, Thomas Kuner
Pubmed Id:https://pubmed.ncbi.nlm.nih.gov/29615726
Parent Title (English):Scientific reports
Publisher:Macmillan Publishers Limited, part of Springer Nature
Place of publication:[London]
Document Type:Article
Year of Completion:2018
Date of first Publication:2018/04/03
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2019/10/16
Tag:Single-molecule biophysics; Super-resolution microscopy
Issue:1, Art. 5507
Page Number:7
First Page:1
Last Page:7
Open Access: This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Institutes:Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Wissenschaftliche Zentren und koordinierte Programme / Sonderforschungsbereiche / Forschungskollegs
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
Licence (German):License LogoCreative Commons - Namensnennung 4.0