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

Abstract

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.

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

“…We found an increased binding affinity (decreased K D ) in the case of Cy3B-P1-Cy3B relative to P1-Cy3B, and a slightly decreased affinity in the case of both SiR-P1-SiR and TMR-P1-TMR relative to P1-SiR and P1-TMR, respectively (Figure S4). We contextualized K D with the measured fluorogenicity and included the previously published value for ATTO 655-P1-ATTO 655 9 (Figure 1C). From this, ATTO 655 and Cy3B exhibit a decreased K D (increase in binding affinity) when dual-labeled to P1 imager strand whereas SiR and TMR show the contrary.…”

“…To address these challenges, FRET-quenched fluorophores attached to extended DNA oligonucleotides were introduced 8 . Recently, DNA oligonucleotides labeled with two identical oxazine fluorophores on either end were shown to exhibit self-quenching through short-distance molecular interactions 9,10 . These probes led to a lower background signal in the unbound state, and consequently to a higher signal-to-background (SBR) ratio.…”

“…These probes led to a lower background signal in the unbound state, and consequently to a higher signal-to-background (SBR) ratio. In addition, an almost two-fold higher photon yield in the bound state increased the localization precision (DNA-PAINT) and spatial resolution (DNA-PAINT and STED-PAINT) 9 . Here, we generalize this approach and introduce new fluorophores to exploit self-quenching with short DNA oligonucleotides.…”

Decoding the molecular interplay of endogenous CD20 and Rituximab with fast volumetric nanoscopy

“…Most recently, self-quenched imager probes labeled with two ATTO 655 dyes were used to demonstrate STED microscopy and DNA-PAINT with reduced background contribution from unbound imager strands at slightly higher photon budget 7 . However, here by using an oxazine dye with superior H-dimer formation tendency 6,8 we could successfully tackle the main problem of DNA-PAINT imaging, which is long acquisition times.…”

“…Lately, efforts have been made to engineer smarter fluorogenic imager probes in order to improve DNA-PAINT imaging. In particular, two main classes of fluorogenic probes have been reported: a) probes end-labeled with a dye and a quencher molecule so that the fluorescence signal of the emitter is efficiently quenched via energy transfer 4 or dye-quencher contact in the unbound state 5 to enable faster imaging at higher probe concentrations and b) probes end-labeled with two identical dyes which form non-fluorescent dimers in the unbound state 6,7 . The non-fluorescent dimer strategy has been used very recently to demonstrate stimulated emission depletion and super-resolution localization microscopy with increased spatial resolution 7 .…”