Super-resolution microscopy techniques have started to transform biological research in many areas. But these methods “are either hard to implement from the reagent side or complex to implement from the equipment side,” explains Ralf Jungmann from the Ludwig Maximilian University Munich and the Max Planck Institute of Biochemistry in Martinsried, Germany. He and his colleagues published a combination of spinning-disk confocal (SDC) microscopy and DNA points accumulation for imaging in nanoscale topography (DNA-PAINT), which makes super-resolution microscopy easy to implement with commonly available microscopes.

Single-molecule localization microscopy (SMLM) methods such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) offer low-nanometer resolution and rely on stochastic switching of fixed and target-bound fluorescent labels. In contrast, PAINT-based methods use freely diffusing probes that bind statically or transiently to the target of interest.

A high signal-to-noise ratio (SNR) is crucial in SMLM for the detection of single-molecule fluorescent events. Selective plane-illumination approaches such as total internal reflection (TIRF) microscopy provide high SNR, but have limited penetration depths. As an alternative to TIRF microscopy, confocal microscopes offer optical sectioning and high SNR by blocking out-of-focus light using a pinhole. SDC microscopes are a variant of confocal microscopes that contain a spinning disk with an array of pinholes. They combine the advantage of high SNR with high-speed acquisition and the use of cameras as spatial detectors.

SDC-PAINT image of Alpha-Tubulin (green), TOM20 (red), and HSP60 (blue). Image adapted with permission from Schueder et al. (Springer Nature).

SDC microscopes have already been applied to perform SMLM experiments using PALM and STORM. However, excitation intensity and detection efficiency are suboptimal in SDC microscopy, which affects the achievable resolution and image quality in SMLM experiments. These “difficulties are not a problem for the DNA-PAINT technique, because the switching between bright and dark [states] is mediated by DNA hybridization reactions,” Jungmann mentions. This decoupling of “switching” from the photophysical properties of the dye molecules, “combined with the very bright and photostable organic dyes that can be used for [DNA-PAINT], basically allowed us to implement [DNA-PAINT] on confocal hardware,” Ralf Jungmann continues.

After evaluating the resolution of their system with DNA origami structures, the researchers demonstrated the capabilities of SDC microscopy in combination with DNA-PAINT (SDC-PAINT) on a range of biological targets. The researchers showed that SDC-PAINT is capable of resolving filamentous microtubule structures alongside the outer membrane and inner matrix in mitochondria throughout the whole cell.

To enable 3D super-resolution imaging with SDC-PAINT, the one modification necessary to their off-the-shelf SDC microscope “was using a cylindrical lens in front of the camera to give us optical astigmatism for point-spread-function engineering [to allow] three-dimensional [super]-resolution imaging,” says Ralf Jungmann. The point-spread-function shaping allowed the researchers to visualize proteins located at the outer mitochondrial membrane and in the mitochondrial matrix. Finally, the authors demonstrate the versatility of SDC-PAINT by visualizing not only proteins, but also RNA and DNA.