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Cell Chem Biol. 2019 Apr 18;26(4):584-592.e6. doi: 10.1016/j.chembiol.2019.01.003. Epub 2019 Feb 7.

Labeling Strategies Matter for Super-Resolution Microscopy: A Comparison between HaloTags and SNAP-tags.

Author information

1
Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, USA; Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT, USA.
2
Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, USA.
3
Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
4
Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT, USA.
5
Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, USA; Department of Biomedical Engineering, Yale University, 55 Prospect Street, New Haven, CT, USA.
6
Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, USA. Electronic address: derek.toomre@yale.edu.

Abstract

Super-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. Organic fluorophores may help here as they can yield more photons-by orders of magnitude-than fluorescent proteins. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAP-tags being the most common. However, how these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. Herein, we compare the two labeling approaches in confocal and STED imaging using various proteins and two model systems. Strikingly, we find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging.

KEYWORDS:

HaloTag; SNAP-tag; STED; fluorophores; live-cell imaging; microscopy; nanoscopy; self-labeling proteins; super-resolution microscopy

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