DNA-PAINT is a technique to enable single-molecule localization through the binding of short (<10 nucleotides) oligonucleotides labeled with a fluorophore to a complementary oligonucleotide bound to a target molecule, typically an antibody or nanobody. The binding of the short oligos is transient in nature, and thus creates a blinking effect akin to dSTORM or PALM. DNA-PAINT has numerous advantages over other blinking techniques:
DNA-PAINT works through the transient binding of a short “imaging oligonucleotide” containing a fluorophore to a complementary oligonucleotide called the “docking strand” on the target of interest such as an antibody, nanobody, aptamer or suicide enzyme ligand. The sample is labeled with the “docking strand” through conventional techniques and prepared for imaging. For imaging, the sample is bathed in imaging buffer (typically PBS but can include oxygen scavengers) and a low (typically 0.1-1 nM) concentration of imaging oligo complementary to the docking strand. The imaging oligo is typically 9-10 nucleotides in length and contains a fluorophore. We recommend Cy3B for DNA-PAINT due to its fluorogenicity and thus lower background. Once in imaging buffer the sample can be imaged. The transient binding of the imaging strand to the docking strand stops the diffusion of the fluorophore allowing it to be imaged on the camera. Since the sample is bathed in a large excess of constantly exchanging imaging strand, the target is essentially unbleachable, making large number of frames and extended Z-stacks possible.
DNA-PAINT allows sub-10 nm localization precision, making it one of the highest precision microscope techniques available. To the right we show a DNA-PAINT experiment performed on the Vutara VXL microscope with a water immersion 1.2 NA objective. The image shows a whole BS-C-1 cell’s tubulin network labeled with secondary antibodies conjugated to a DNA-PAINT secondary antibody. The inset to the left shows a zoomed in section of the tubulin network. The lumen of the microtubule is clearly visible.
Sample: BS-C-1 labeled with anti-tubulin. Secondary DNA-PAINT antibodies were purchased from Massive-Photonics.com.
DNA-PAINT has enormous potential for multiplexed imaging using the Vutara and integrated fluidics unit. Using orthogonal docking strands on different probes, a potentially unlimited number of targets are possible. To the left we show a two-color DNA-PAINT experiment performed on the Vutara VXL single-molecule localization microscope. Tubulin is labeled in cyan and clathrin in magenta. Furthermore, due to the unbleachable nature of DNA-PAINT large Z-stacks are possible (bottom).
Sample: BS-C-1 labeled with anti-tubulin and anti-clathrin. Secondary DNA-PAINT antibodies were purchased from Massive-Photonics.com.
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