Super-resolution microscopy with DNA-PAINT

Abstract

Super-resolution techniques have begun to transform biological and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resolution microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resolution microscopy. Using the programmability and specificity of DNA molecules as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resolution techniques, making them compatible with virtually any single-molecule-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise molecule counting and ultra-high, molecular-scale (sub-5-nm) spatial resolution, reaching ∼1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample preparation, multiplexed data acquisition, data simulation, super-resolution image reconstruction and post-processing such as drift correction, molecule counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1-2 d.

Bacterially Derived Antibody Binders as Small Adapters for DNA‐PAINT Microscopy

“…Different methods have been introduced to conjugate DNA docking strands to protein‐based affinity reagents. Typically, a bifunctional chemical crosslinker harboring a reactive moiety (which can subsequently react with a modified DNA oligonucleotide) was used to react with amino groups or reduced thiols . To produce DNA‐modified secondary labeling reagents, protein A from Staphylococcus aureus and protein G from Streptococcus sp.…”

Nucleic‐Acid Structures as Intracellular Probes for Live Cells

“…Strauss et al developed slow off‐rate modified aptamers (SOMAmers) for monitoring cell surface and intracellular targets using DNA points accumulation in nanoscale topography (DNA‐PAINT) . DNA‐PAINT is a single molecule imaging technique that allows ≈5 nm spatial resolution . The method involves the binding of dye‐labeled DNA strands to their complements transiently.…”

Versatile multiplexed super-resolution imaging of nanostructures by Quencher-Exchange-PAINT

“…Binding-event rate optimization is crucial for efficient DNA-PAINT imaging [15,16]. If too many binding events per frame are observed, the risk of overlapping events increases, reducing the localization precision.…”

“…This arrangement is typically achieved either with specially designed chambers [16], which can require complex preparation, or with multiple washing steps in an open-top chamber. A drawback when working with an open chamber is that accidental full draining can deteriorate sample quality or dislodge the sample.…”

“…Because the fluorescent marker is not fixed on the target structure, multiplexed imaging can be achieved by exchanging imager solutions with different sequences, a method known as Exchange-PAINT [14,16,17]. This imaging of multiple targets with Exchange-PAINT by means of the same fluorescent dye gives an image free of chromatic aberrations.…”