Fluorescence microscopy with Ångström resolution

Cells, the basic units of life, contain a multitude of complex structures, processes and mechanisms that maintain and perpetuate living systems. Many core cellular elements such as DNA, RNA, proteins and lipids are only a few nanometres in size. This makes them considerably smaller than the resolution limit of conventional light microscopy. The exact composition and arrangement of these molecules and structures is therefore often unknown, which leads to a lack of mechanistic understanding of fundamental aspects of biology.

In recent years, so-called super-resolution techniques have made enormous progress and make it possible to resolve subcellular structures below the classical diffraction limit of light. Single-molecule localisation microscopy (SMLM) is a technique that allows structures in the order of ten nanometres to be resolved by separating their individual fluorescence emissions in time. Since individual target molecules light up stochastically (they blink) in an otherwise dark field of view, their positions can be determined with an accuracy below the diffraction limit. DNA-PAINT, invented by the Jungmann group, is an SMLM technique that uses temporary hybridisation of dye-labelled DNA "imager" strands to achieve the necessary blinking for super-resolution. So far, however, even DNA-PAINT has not been able to resolve the smallest cellular structures.

Unlimited spatial resolution

In the current study, which was led by first authors Susanne Reinhardt, Luciano Masullo, Isabelle Baudrexel and Philipp Steen together with Jungmann, the team presents a new approach in super-resolution microscopy that enables fundamentally "unlimited" spatial resolution. The new technique, called "Resolution Enhancement by Sequential Imaging", or RESI for short, utilises the ability of DNA-PAINT to encode the identity of target objects using unique DNA sequences. Molecules in close proximity to each other that cannot be resolved with classical SMLM are labelled with different DNA sequences. This creates an additional distinguishing feature. By sequentially imaging first one and then the other sequence (and thus molecule), they can now be clearly separated and resolved. As they are imaged one after the other, the targets can be arbitrarily close to each other, which is not possible with any other technique. Furthermore, RESI does not require specialised microscopes, it can be used with any standard fluorescence microscope, making it easily accessible to almost all researchers.

To demonstrate the resolution enhancement of RESI, the team took on the challenge of resolving one of the smallest spatial distances in a biological system: The distance between individual bases along a DNA double helix that are less than one nanometre (one billionth of a metre) apart. In a DNA origami nanostructure, which contained single-stranded DNA sequences only one base pair apart, the research team was able to measure a distance of 0.85 nm (or 8.5 nanometres) between neighbouring bases. The researchers achieved this measurement with a precision of 1 Ångström, or one ten-billionth of a metre, which underlines the unprecedented capabilities of the RESI method.

Mechanism of action of rituximab

It is important that the technique is universal and not just limited to applications in DNA nanostructures. To this end, the team investigated the molecular mechanism of action of rituximab, an anti-CD20 monoclonal antibody that was first approved for the treatment of CD20-positive blood cancer in 1997. However, studying the effects of such drug molecules on molecular receptor patterns is beyond the scope of conventional microscopy techniques. Understanding whether and how such patterns change during disease and treatment is of great importance not only for basic mechanistic research, but also for the development of new targeted disease therapies. With RESI, Jungmann and his team were able to reveal the natural arrangement of CD20 receptors in untreated cells as dimers and uncover how CD20 reorganised into chains of dimers upon drug treatment. The findings at the single protein level now help to better understand the molecular mode of action of rituximab.

Since RESI is carried out in whole, intact cells, the technique closes the gap between purely structural methods such as X-ray crystallography or cryogenic electron microscopy and conventional imaging methods with lower resolution for whole cells. Jungmann and his team are convinced that "this unprecedented technique is a real game changer not only for super-resolution, but also for biological research as a whole".

Source: Max Planck Society from 24 May 2023