DNA-PAINT

DNA-PAINT (Point Accumulation In Nanoscale Topography)

DNA-PAINT allows for <10 nm localization precision in single molecule localization microscopy

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:

  • Higher photon yield. The blinks are typically longer lasting than in conventional dSTORM resulting in higher photon yields from the fluorophore, therefore allowing for a much higher localization precision (<10 nm) when compared to such methods as dSTORM and PALM.
  • The imaging is practically unbleachable. The sample is bathed in an excess of fluorophore, allowing for extremely long-lasting imaging.
  • Unlimited multiplexing potential. Since target specificity is set by nucleotide sequence, multiple targets can be labeled with different oligo sequences. The Vutara’s microfluidics unit can be utilized such that the imaging strand for a given target can be washed from the sample and different imaging strands labeling different biological targets can be added.

DNA-PAINT: How Does the Technique Work?

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 How it works
The cartoon above shows how DNA-PAINT works. The target protein (tubulin) is labeled with an antibody labeled with the docking strand oligo. The sample is then bathed in imaging strand oligos. The transient binding of the fluorescently labeled imaging strand to the docking strand causes the sample to appear to blink, which can then be localized in the Vutara SRX software.

DNA-PAINT: High Precision Localization Microscopy

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 352 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 one color with zoomed section

DNA-PAINT – Multicolor Imaging

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 right we show a two-color DNA-PAINT experiment performed on the Vutara 352 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

Two color DNA PAINT experiment performed on the Vutara 352
BS C 1 Labeled

DNA-PAINT – Further Reading

Jungmann R, Steinhauser C, Scheible M, Kuzyk A, Tinnefeld P, Simmel FC. Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami. Nano Letters. 2010 Oct;10(11):4756-4761.

 

Jungmann R, Avendaño MS, Woehrstein JB, Dai M, Shih WM, Yin P. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nature Methods. 2014 Mar;11(3):313–8.

 

Schueder F, Strauss MT,…Jungmann R et al. Universal Super-Resolution Multiplexing by DNA Exchange. Angew. Chem. Int. Engl. 2017 Mar;56:4052-4055.

 

Lin D, Gagnon LA, Howard MD, Halpern AR, Vaughan JC. Extended-Depth 3D Super-Resolution Imaging Using Probe-Refresh STORM. Biophysical J. 2018 April;114(8):1980-1987.