Protein‐Specific, Multicolor and 3D STED Imaging in Cells with DNA‐Labeled Antibodies

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.

DNA hybridisation kinetics using single-molecule fluorescence imaging

“…Deoxyribonucleic acid (DNA) hybridisation, especially of short DNAs, is an essential process in biology, however, much is still unknown about the exact process of hybridisation and its kinetics. As DNA hybridisation is also used across a range of biological and biotechnological applications such as hybridisation-based next-generation DNA sequencing [1], fluorescence-based in situ imaging [2], and super-resolution imaging [3,4], it is essential to achieve a much more complete understanding of the kinetics of hybridisation.…”

“…DNA-PAINT uses repeated transient hybridisation of short fluorescently labelled DNA to an immobilised complementary DNA to create a super-resolved image, see Figure 1D, with the method being quantitative [10] and having the ability to create multicolour images [11,12], even to create 124-plex images within minutes [13]. Since its invention, DNA-PAINT has been implemented alongside many different super-resolution microscopy techniques in order to image targets inside cells, such as structured illumination microscopy (SIM) [14], stimulated emission depletion (STED) microscopy [4], stochastic optical reconstruction microscopy (STORM) [15,16] and spinning disk confocal (SDC) microscopy [17]. DNA-PAINT so far has a wide range of biological applications such as imaging synaptic proteins [18], imaging forces inside live cells [19] (Figure 1E), creating 3D images of internal cell structures [11,16] and immunostaining of neuronal cells, tissues and microtubules [4,14] (Figure 1D).…”

“…Since its invention, DNA-PAINT has been implemented alongside many different super-resolution microscopy techniques in order to image targets inside cells, such as structured illumination microscopy (SIM) [14], stimulated emission depletion (STED) microscopy [4], stochastic optical reconstruction microscopy (STORM) [15,16] and spinning disk confocal (SDC) microscopy [17]. DNA-PAINT so far has a wide range of biological applications such as imaging synaptic proteins [18], imaging forces inside live cells [19] (Figure 1E), creating 3D images of internal cell structures [11,16] and immunostaining of neuronal cells, tissues and microtubules [4,14] (Figure 1D). For DNA-PAINT, and other biotechnologies, a greater understanding of the kinetics of DNA hybridisation will lead to the optimisation of hybridisation assays making these technologies more powerful.…”

Aptamers with Tunable Affinity Enable Single‐Molecule Tracking and Localization of Membrane Receptors on Living Cancer Cells

“…Photobleached labels thus remain tightly associated to their target receptors, which in turn cannot be further imaged. DNA duplexes with programmable hybridization kinetics were successfully exploited to promote faster exchange of fluorescent reporters . However, this strategy still requires a high affinity probe to label target receptors and expose a DNA docking strand for the hybridization with dye‐conjugated DNA strands diffusing in solution.…”

Multi‐Color, Bleaching‐Resistant Super‐Resolution Optical Fluctuation Imaging with Oligonucleotide‐Based Exchangeable Fluorophores

“…As such, these labels are insensitive to common photobleaching and thus yield a constant fluorescence signal over time, which has been successfully exploited in SMLM [3,4,11] and stimulated emission depletion (STED) microscopy. [12] In order to implement exchangeable labels for SOFI, we built on the concept of DNA-PAINT, in which stochastic "blinking" is achieved by transient and reversible binding of exchangeable fluorophore-labeled oligonucleotides (imager strands) from a buffer reservoir to complementary DNAsequences (docking strands). Using secondary antibodies for covalent attachment of docking strands (Figure 1 A) allows highly specific and multiplexed imaging of cellular structures with high spatial resolution.…”