Alexander Auer & Ralf Jungmann discuss the use of DNA-PAINT, a super-resolution microscopy technique
Super-resolution1 techniques have enabled researchers to perform imaging below the classical diffraction-limit of light with thus far unprecedented precision. In single-molecule localisation implementations, molecules are ‘switched’ between non-fluorescent dark- (or off-) and fluorescent bright-states (or on-states) to assign and detect their position with sub-diffraction precision. By acquiring not only one, but rather a whole stack of images, in which only a random subset of fluorescent molecules (which differ from frame to frame) are recorded in the “on-state”, a super-resolved image can be constructed.
The recently introduced super-resolution method DNA-point accumulation for imaging in nanoscale topography (PAINT)2 is based on transient DNA-DNA interactions. Compared to other stochastic approaches, such as STORM3, PALM4, or GSDIM5 the fluorescence molecules aren’t switched between dark and bright states, but the so-called “blinking” in DNA-PAINT is created by transient hybridisation (binding and unbinding) of short fluorescent DNA strands (imagers) to their targets (Fig. 1a). Once the imager is bound to the docking site, the fluorophore is immobilised and can be detected by a camera. DNA-PAINT does not suffer from photobleaching as the imager strands are dynamically replenished. This allows the extraction of the full photon capacity of an immobilised imager strand upon binding to the docking site, facilitating superior resolution down to a few nanometres6.
The high signal-to-background ratio enables the combination of DNA-PAINT and confocal approaches, such as spinning disc confocal microscopy7.
The team at PCO assayed a new non-cooled scientific CMOS camera (pco.panda 4.2) with synthetic DNA nanostructures. Therefore, they decorated a flat rectangle DNA origami with DNA-PAINT docking sites in a grid pattern with 10nm spacing (Fig. 1b) and imaged the surface-bound structures using total internal reflection fluorescence8 (TIRF) microscopy. Individual docking sites could be localised with a precision of around 1.5nm, translating to a remarkable FWHM-resolution ~3.6nm (Fig. 1c).
Due to the programmable nature of DNA interactions in DNA-PAINT, multicolour image acquisition is not limited to spectral multiplexing. The DNA sequence of the imager strand can serve as a pseudo-colour, where every orthogonal sequence represents a unique colour. This enables multiplexed imaging using the same best-performing fluorescent dye, in a method called exchange-PAINT9. Image acquisition is then performed sequentially. The transiently binding imager strands can be removed using washing buffer and the new ‘colour’ (e.g. imagers with a different sequence) can be introduced.
APPLICATION DNA-PAINT
To image cellular components with DNA-PAINT, labelling probes (for example antibodies10, nanobodies11 or affimers12) were conjugated with short DNA oligonucleotides serving as the docking site. The team investigated the imaging capability of a non-cooled sCMOS camera in a cellular environment of a fixed COS7 cell, where alpha tubulin was labeled with primary and secondary antibody (Fig.2). The overview (Fig. 2a) shows the super-resolved microtubule network. Comparison of the diffraction-limited zoom-in panel in Fig. 2b with the super-resolved zoom-in in Fig. 2c clearly displays the increase in resolution, allowing the discrimination and the distance measurement (Fig. 2d) between individual tubulin filaments. Clearly a non-cooled sCMOS camera is well suited to do these kind of super resolution measurements with the DNA-PAINT method.
REFERENCES
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3 Rust, M.J., M. Bates, and X.W. Zhuang, Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods, 2006. 3(10): p. 793-795.
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10 Schnitzbauer, J., et al., Super-resolution microscopy with DNA-PAINT. Nat Protoc, 2017. 12(6): p. 1198-1228.
11 Agasti, S.S., et al., DNA-barcoded labelling probes for highly multiplexed Exchange-PAINT imaging. Chem Sci, 2017. 8(4): p. 3080-3091.
12 Schlichthaerle, T., et al., Site-specii c labeling of Afi mers for DNA-PAINT microscopy. Angew Chem Int Ed Engl, 2018.
Alexander Auer & Ralf Jungmann wotk with PCO cameras
