DNA-PAINT operates on the principle of transient binding between complementary DNA strands to achieve super-resolution imaging. A target molecule, such as a protein, is first labeled with a docking strand of single-stranded DNA (ssDNA), which is immobilized onto the target via a specific antibody or aptamer. Meanwhile, an imager strand, also composed of ssDNA and linked to a fluorescent dye, is kept in solution.

When excited by a laser, the imager strand binds to the docking strand, resulting in a fluorescent “blink” that can be captured by a high-resolution camera. These rapid binding and unbinding events are recorded over time, allowing advanced algorithms to reconstruct precise images at resolutions of 10-30 nm. This process effectively circumvents the limitations of optical diffraction, enabling detailed visualization of subcellular structures.

Significant advantages in imaging compared to traditional fluorescence techniques are offered through DNA-PAINT. Its unique labeling strategy allows for precise targeting and high localization accuracy, while the continuous replenishment of fluorophores prevents photobleaching, a common issue in other methods. The technique also supports multiplexing, enabling simultaneous imaging of multiple targets by using distinct docking and imager strands or sequential flow of new imager strands to new targets over time.

Notably, DNA-PAINT has been demonstrated to visualize up to 124 colors in a single sample through innovative kinetic fingerprinting techniques. Additionally, the quantitative capabilities of DNA-PAINT (i.e. qPAINT) facilitates molecular counting and quantitation, a key aspect to what makes super-resolution microscopy, and more notably DNA-PAINT, a valuable technique.

DNA-PAINT is compatible with various super-resolution microscopy platforms, including the Nanoimager, which is particularly suited for capturing the rapid dynamics of binding events. This versatility allows researchers to integrate DNA-PAINT with other single-molecule localization microscopy (SMLM) techniques, such as dSTORM or PALM, enabling multimodal imaging strategies. Furthermore, the Nanoimager’s microfluidics capabilities streamline the process of exchanging imaging strands, facilitating automated and efficient experiments. Beyond DNA, the platform can accommodate transient interactions involving proteins and glycan-lectin systems, broadening the scope of applications in diverse research fields.