Self‐quenched Fluorophore Dimers for DNA‐PAINT and STED Microscopy

Super‐resolution techniques like single‐molecule localisation microscopy (SMLM) and stimulated emission depletion (STED) microscopy have been extended by the use of non‐covalent, weak affinity‐based transient labelling systems. DNA‐based hybrid systems are a prominent example among these transient l...

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Published in:Angewandte Chemie International Edition 2023-09, Vol.62 (39), p.e202307538-n/a
Main Authors: Kessler, Laurell F., Balakrishnan, Ashwin, Deußner‐Helfmann, Nina S., Li, Yunqing, Mantel, Maximilian, Glogger, Marius, Barth, Hans‐Dieter, Dietz, Marina S., Heilemann, Mike
Format: Article
Language:English
Subjects:
Chemical compounds
Deoxyribonucleic acid
Dimers
DNA
DNA-PAINT
Fluorescence
Fluorescence Correlation Spectroscopy
Fluorescence Quenching
Fluorophores
Hybrid systems
Labeling
Microscopy
Oligonucleotides
Optical properties
Single-Molecule Localization Microscopy
Spatial discrimination
Spatial resolution
Stimulated emission
Stimulated Emission Depletion
Thermodynamic properties
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Summary:Super‐resolution techniques like single‐molecule localisation microscopy (SMLM) and stimulated emission depletion (STED) microscopy have been extended by the use of non‐covalent, weak affinity‐based transient labelling systems. DNA‐based hybrid systems are a prominent example among these transient labelling systems, offering excellent opportunities for multi‐target fluorescence imaging. However, these techniques suffer from higher background relative to covalently bound fluorophores, originating from unbound fluorophore‐labelled single‐stranded oligonucleotides. Here, we introduce short‐distance self‐quenching in fluorophore dimers as an efficient mechanism to reduce background fluorescence signal, while at the same time increasing the photon budget in the bound state by almost 2‐fold. We characterise the optical and thermodynamic properties of fluorophore‐dimer single‐stranded DNA, and show super‐resolution imaging applications with STED and SMLM with increased spatial resolution and reduced background. A new approach for fluorophore‐labelled DNA oligonucleotides is introduced that relies on short‐distance self‐quenching of two identical fluorophores. This strategy achieves a reduction in background fluorescence, while at the same time increasing the photon budget two‐fold. These probes can be applied to super‐resolution techniques such as stimulated emission depletion (STED) and single‐molecule localisation microscopy (SMLM).
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.202307538