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

Abstract

The optical super-resolution technique DNA-PAINT (Point Accumulation Imaging in Nanoscale Topography) provides a flexible way to achieve imaging of nanoscale structures at ~10-nanometer resolution. In DNA-PAINT, fluorescently labeled DNA "imager" strands bind transiently and with high specificity to complementary target "docking" strands anchored to the structure of interest. The localization of single binding events enables the assembly of a superresolution image, and this approach effectively circumvents photobleaching. The solution exchange of imager strands is the basis of Exchange-PAINT, which enables multiplexed imaging that avoids chromatic aberrations. Fluid exchange during imaging typically requires specialized chambers or washes, which can disturb the sample. Additionally, diffusional washout of imager strands is slow in thick samples such as biological tissue slices. Here, we introduce QuencherExchange-PAINT-a new approach to Exchange-PAINT in regular open-top imaging chambers-which overcomes the comparatively slow imager strand switching via diffusional imager washout. Quencher-Exchange-PAINT uses "quencher" strands, i.e., oligonucleotides that prevent the imager from binding to the targets, to rapidly reduce unwanted single-stranded imager concentrations to negligible levels, decoupled from the absolute imager concentration. The quencher strands contain an effective dye quencher that reduces the fluorescence of quenched imager strands to negligible levels. We characterized QuencherExchange-PAINT when applied to synthetic, cellular, and thick tissue samples. Quencher-Exchange-PAINT opens the way for efficient multiplexed imaging of complex nanostructures, e.g., in thick tissues, without the need for washing steps.

Repeat DNA-PAINT suppresses background and non-specific signals in optical nanoscopy

“…Streptavidin-functionalized polystyrene particles with a diameter of 500 nm (Microparticles GmbH, Germany) were labeled with biotinylated oligonucleotides (Fig. 1a: docking motifs 1x RD, 3x RD, and 6x RD, see Supplementary Table 1) as described elsewhere 24 . Briefly the microspheres were dispersed in TE buffer containing 300 mM NaCl and the docking strands in 4x excess concentration as compared to the binding capacity of the beads.…”

Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy

“…The very high affinity of biotin ensures an effectively permanent attachment of biotinylated S1 and S2 constructs. Streptavidin was attached to the surface of polystyrene colloidal particles, to provide a convenient imaging geometry as previously used in the context of DNA-PAINT by ourselves 34 and others 35 . Biotinylated S1 and S2 constructs were mixed thoroughly before streptavidin-coated particles were dispersed on the coverslip to ensure a stochastic attachment of S1 and S2 to the biotin-binding sites.…”

3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples

“…Now, commercial slides with 3D DNA-origami structures are available which provide a stringent test of 3D imaging performance. In addition, we [45] and others [46,47] have established DNA-PAINT samples based on surface coated microspheres which can be used for a variety of testing purposes associated with DNA-PAINT, including their use as convenient 3D structures of known shape.…”

“…6B) was generated by simulation using a Gaussian PSF model, as recording a widefield image is impractical due to the fact that fluorochromes were only attached to the imagers which diffuse freely in the solution. In practice, an additional fluorochrome with different emission spectrum from the imager, for example, a Cy3 dye, can be appended to each docking site to produce continuous fluorescence for labelling checks under widefield illumination [45]. The drift-corrected cloud of localisation ( Fig.…”

“…Oligonucleotide sequences were adapted from [65], imager: CTAGATGTAT -3'Atto 655, docking: 5'Biotin -TTATACATCTA. Microsphere labelling and imaging as described elsewhere [45]. Docking strand oligonucleotides were attached to streptavidin-coated polystyrene colloidal particles (r = 250 nm, binding capacity 500 pmole of biotin per mg of particles, Microparticles GmbH) by suspension of docking strands at 4x excess (1 µM) in TE buffer containing the polystyrene particles (0.5 mg/ml) and 300 mM NaCl.…”