An image of a blob titled CD20 receptors with the top half labelled Diffraction limited and ispixellated in grey scale and the bottom labelled DNA-PAINT is sharper in red and black. There is a 5 micrometre scale in the corner.

Source: © Reinhardt et al, Nature 2023

The new Resi microscopy technique can deliver vastly improved resolution, even over super resolution microscopy. Here a CD20 receptor is shown using ordinary light microscopy (top) and Resi

An optical-microscopy technique has been created that can distinguish between tiny objects less than 1nm apart. The technique could enable living cells to be viewed in greater detail than ever before.

The new method, dubbed resolution enhancement by sequential imaging (Resi), means the resolving power of fluorescent microscopy can now operate in the Ångström range for the first time. Conventional super-resolution microscopy can distinguish between objects between 15 to 20nm apart, but this does not directly translate to experiments in cells. When super-resolution microscopy came along it was so revolutionary in the life sciences that its developers won the chemistry Nobel prize in 2013.

In contrast to existing techniques, Resi involves labelling adjacent copies of the same target molecule with a different tag. The researchers showed that when each of the tags was lit up, they could be clearly differentiated, even if closely spaced together, by imaging them sequentially.

By collecting multiple signals from each tagged molecule, the average position of each set could be determined with a higher level of precision than is possible with a single tagged molecule, to obtain a ‘super-super resolution’ image.

Using this technique, the researchers showed that Resi could distinguish between two tags just a few atoms, or less than a nanometre, apart in an engineered structure made of DNA. They were also able to label and image CD20, a protein targeted by the anticancer drug rituximab. By mapping the molecular arrangement of CD20 in situ in untreated and drug-treated cells, Resi was able to reveal new information about how the protein is arranged and how that arrangement is affected by binding by the drug.

A diagram showing two stacks. Both have a lower of 2 molecule positions. The first stack has a top layer with many overlapping spots. The second stack has 2 middle layers detecting one of each of the molecules so its top layer shows each average position

Source: © 2023 Springer Nature Limited

In a group of techniques known as localisation microscopy (left), all the copies of the molecule of interest are labelled with tags that light up (blink) with fluorescence, a few at a time. Over many repetitions, the positions of many of the molecules can be found from the average positions of the blinks (each blink is shown as a grey dot). However, blinks from tagged molecules within about 10 to 20nm of each other overlap, making it difficult to distinguish the individual molecules. In Resi (right), adjacent copies of the same target molecule are each labelled with a different tag, enabling the blinks from closely spaced molecules to be differentiated by imaging them in different time periods. Tagged molecules less than 1nm apart can be distinguished

Using Resi, the researchers were able to resolve more than 2000 DNA origami structures and, separately, 1000 nuclear pore complex proteins in areas of around 67µmin 100 minutes. This, they added, made the technique applicable as a ‘sufficiently high-throughput tool’ for cell biology.

However, a limitation with the method was that structures observed by Resi must stay still long enough to gather precise information. For some biological experiments this means that cells must be ‘fixed’, which can introduce structural distortion and prevent dynamic processes from being visualised over time.

The size of the tag is also a limiting factor because it is the position of the fluorescent part of a tag that is observed, rather than the molecule of interest that the tag is connected to.

An oval made up of tiny rings shown in an inset on a 500 nm scale. The full image has a 5 micrometre scale and is divided into a Diffraction limited area which is greyscale and blurry. The lower portion is labelled DNA-PAINT is black and red and sharp

Source: © Reinhardt et al, Nature 2023

Nuclear pore complexes shown using conventional light microscopy (top) and Resi, demonstrating the exceptional resolution of the proteins that can be achieved

Despite this, the researchers said that Resi could be used as a pre-screening method for personalised treatments and could be a tool in drug design.

‘Resi is … poised to close the gap between 3D fluorescence super-resolution microscopy in whole intact cells and cryo-EM structural studies of individual supramolecular complexes, introducing a paradigm shift in fluorescence imaging by pushing optical microscopy to Ångström resolutions.’

‘We are very excited about the development of Resi, which – for the first time – allows researchers to resolve sub-nanometre distances using off-the-shelf instrumentation,’ says Ralf Jungmann, a group leader at the Max Planck Institute of Biochemistry and one of the authors of the study. ‘It is a paradigm shift for optical microscopy, enabling intramolecular imaging and closing the gap between super-resolution microscopy and structural biology studies. Resi is poised to deliver information key to understanding complex biological systems.’

Xiaolin Nan, assistant professor of biomedical engineering at Oregon Health and Science University, tells Chemistry World that Resi is ‘very exciting’. ‘I believe the method represents a major advance in biological imaging with significant implications in many areas of bioscience including structural biology,’ he adds.