Point Accumulation in Nanoscale Topography
Point Accumulation in Nanoscale Toppography, PAINT, was initially developed as a dye-based strategy for localization microscopy (ref NileRed), by exploiting fluorescent molecules that bind transiently to a target. Fast binding and unbinding generate a sequential single-molecule signal (“blinking”): this can be captured over time, and the position of each emitter localized with high precision using strategies analogous to those used in dSTORM and PALM.
DNA-PAINT PRINCIPLE
In DNA-PAINT the transient association of the fluorophore to a target molecule is mediated by the pairing of short (<10 nucleotides) complementary DNA sequences:
- the « docking» strand is coupled to the target molecule, usually through an antibody, nanobody, aptamer or other high affinity probe
- the « imager» strand carries the fluorophore, and it is free to diffuse in the imaging buffer
Upon DNA hybridization the fluorophore is transiently immobilized near the target moecule, and thus excited by the laser light , tipically in TIRF or HiLO configuration. The emitted light can then be captured by the camera as a diffraction limited flash. By adjusting sequence, concentration, ratio of the DNA strands, and composition of the imaging buffer, at each time point only a few fluorophores will be imaged, enabling stochastic super resolution microscopy.
What are the advantages of DNA PAINT?
- Ultra-high resolution
In a DNA-PAINT experiment the duration of a single binding event (a blink) can be programmed to be much longer than a dSTORM flash (link STORM), yielding a higher number of photons per fluorophore. Consequently a much higher precision of localization can be reached; thus DNA-PAINT can achieve true molecular resolution (1) . Moreover, bleaching is practically non-existent: the sample is imaged within an excess of fluorophore that constitutes a practically inextinguishable reservoir. This allows for very prolonged imaging and the accumulation of extremely dense datasets.
- Quantitative imaging
The hybridization of DNA oligonucleotides is highly predictable and tunable; combined with irrelevant bleaching this permits a very fine control of PAINT imaging and thus accurate quantitative imaging,, or qPAINT (2) i.e. true counting of molecular species.
- Multiplexing
In DNA-PAINT target specificity is determined by the DNA strand sequences; by designing the shortest oligonucleotides with minimal cross-talk (i.e. orthogonal sequences) it is possible to label with different, orthogonal docking strands a very large number of targets, limited only by the availability of affinity probes (antibodies, nanobodies or other) and the accessibility of the biological target. Each given target can then be imaged sequentially, using the appropriate imaging strand and alternating washing steps (3, 4). An advantage of this multiplexing strategy is that the same fluorophore can be used for each target molecule, thus allowing homogeneous precision of localization across biological targets and removing chromatic aberrations in between them.
What are the drawbacks of DNA-PAINT and how to overcome them?
Image acquisition in DNA-PAINT is slow compared to most other approaches; the resulting, very long imaging sessions require often additional efforts for correction of sample drift. Although recent development (ref Jungmann) offer hope that PAINT workflow can be accelerated, low imaging speed remains a major drawback of this super-resolution technique and limits its implementation.
It is possible to combine DNA-PAINT with Abbelight spectral demixing strategy (link), and image simultaneously up to three target molecules, with no chromatic aberrations, thus reducing by two thirds the duration of even the most complex DNA-PAINT workflow.
Moreover, Abbelight homogeneous TIRF/HiLO illumination over large FOV (link) further compensates for DNA-PAINT temporal limitation by permitting the imaging of very large sample regions of interest (~200×200 microns) with the same resolution.
Essential bibliograph
1. A. Sharonov, R. M. Hochstrasser, Proc. Natl. Acad. Sci. U. S. A. 103, 18911–18916 (2006).
2. Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Super-resolution microscopy with DNA- PAINT. Nat. Protoc. 12, 1198–1228 (2017).
3. Jungmann, R. et al. Quantitative super-resolution imaging with qPAINT. Nat. Methods 13, 439–442 (2016).
4. Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313–318 (2014).
5. Agasti, S. S. et al. DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging. Chem. Sci. 8, 3080– 3091 (2017).
6. F. Schueder et al., Nat. Methods. 16, 1101–1104 (2019).