Abstract
Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal‐to‐noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore‐labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein‐specific antibodies. The constant exchange of fluorophore labels in DNA‐based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two‐color STED imaging of whole cells.
Highlights
Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used
We demonstrate confocal and stimulated emission depletion (STED) microscopy with short, fluorophorelabeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies
The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels
Summary
Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels. Microscopy has contributed to our to-date understanding of cell biology.[1,2] As with other fluorescence microscopy techniques that use comparably high laser intensities, photobleaching of the fluorophore labels limits image quality and information content.
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