When Weak Is Strong: A Plea for Low‐Affinity Binders for Optical Microscopy

Albertazzi, Lorenzo; Heilemann, Mike

doi:10.1002/anie.202303390

Cited by 13 publications

(8 citation statements)

References 32 publications

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“…membrane-binding labels 14 . An extension to more weak-affinity fluorophore labels 15 or protein tags 16 and combinations thereof 5 can be envisioned. In conclusion, we show that the concept of self-quenching dimers could be extended to other classes of fluorophores, and that these probes provide a higher SBR and improved spatial resolution in cutting-edge microscopy applications.…”

Section: Discussionmentioning

confidence: 99%

Self-quenched fluorophore-DNA labels for super-resolution fluorescence microscopy

Kessler,

Balakrishnan,

Menche

et al. 2024

Preprint

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Protein labeling through transient and repetitive hybridization of short, fluorophore-labeled DNA oligonucleotides has become widely applied in various optical super-resolution microscopy methods. The main advantages are multi-target imaging and molecular quantification. A challenge is the high background signal originating from the presence of unbound fluorophore-DNA labels in solution. Here, we report self-quenching of fluorophore dimers conjugated to DNA oligonucleotides as a general concept to reduce the fluorescence background. Upon hybridization, the fluorescence signal of both fluorophores is fully restored. Here, we expand the toolbox of fluorophores suitable for self-quenching and report their spectra and hybridization equilibria. We apply self-quenched fluorophore-DNA labels to stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM) and report improved imaging performances.

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Section: Discussionmentioning

confidence: 99%

Self-quenched fluorophore-DNA labels for super-resolution fluorescence microscopy

Kessler,

Balakrishnan,

Menche

et al. 2024

Preprint

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“…This can be accomplished by re-training a new model on the expanded dataset, or through transfer learning on a dataset consisting of the earlier-stage aggregates. Low-affinity binders 61 that could better capture the diversity of misfolded protein aggregates could have great potential to extend our method to enable the identification and label-free imaging of different aggregation states on the pathway to inclusion formation.…”

Section: Discussionmentioning

confidence: 99%

Label-free identification of protein aggregates using deep learning

Ibrahim,

Grußmayer,

Riguet

et al. 2023

Nat Commun

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Protein misfolding and aggregation play central roles in the pathogenesis of various neurodegenerative diseases (NDDs), including Huntington’s disease, which is caused by a genetic mutation in exon 1 of the Huntingtin protein (Httex1). The fluorescent labels commonly used to visualize and monitor the dynamics of protein expression have been shown to alter the biophysical properties of proteins and the final ultrastructure, composition, and toxic properties of the formed aggregates. To overcome this limitation, we present a method for label-free identification of NDD-associated aggregates (LINA). Our approach utilizes deep learning to detect unlabeled and unaltered Httex1 aggregates in living cells from transmitted-light images, without the need for fluorescent labeling. Our models are robust across imaging conditions and on aggregates formed by different constructs of Httex1. LINA enables the dynamic identification of label-free aggregates and measurement of their dry mass and area changes during their growth process, offering high speed, specificity, and simplicity to analyze protein aggregation dynamics and obtain high-fidelity information.

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“…Weak affinity non‐covalent fluorophore labels complement covalent labelling of target proteins and have been applied to various super‐resolution techniques [1] . These exchangeable or renewable labels provide the advantage of constant signal for long acquisition times, and thus minimise photobleaching, as well as offering multiplexing solutions beyond the “spectral barrier” [2–7] .…”

Section: Figurementioning

confidence: 99%

“…Weak affinity non-covalent fluorophore labels complement covalent labelling of target proteins and have been applied to various super-resolution techniques. [1] These exchangeable or renewable labels provide the advantage of constant signal for long acquisition times, and thus minimise photobleaching, as well as offering multiplexing solutions beyond the "spectral barrier". [2][3][4][5][6][7] A widely used concept is DNA hybridisation to achieve transient binding between an imager strand carrying a fluorophore and a sequence complementary docking strand coupled to the target either directly or via an antibody.…”

mentioning

confidence: 99%

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

et al. 2023

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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 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.

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When Weak Is Strong: A Plea for Low‐Affinity Binders for Optical Microscopy

Cited by 13 publications

References 32 publications

Self-quenched fluorophore-DNA labels for super-resolution fluorescence microscopy

Self-quenched fluorophore-DNA labels for super-resolution fluorescence microscopy

Label-free identification of protein aggregates using deep learning

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

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