Since many core components of cells — like DNA, RNA, proteins, and lipids — are just a few nanometers in size and substantially smaller than the resolution limit of traditional light microscopy, the exact composition and arrangement of these molecules and structures is thus often unknown. This results in a lack of mechanistic understanding of fundamental aspects of biology. Drawing on recent improvements to superresolution imaging, including single-molecule localization microscopy, or SMLM, researchers from the Max Planck Institute of Biochemistry and Ludwig-Maximilians-Universität Munich have developed a technique that enhances the resolution of fluorescence microscopy down to the angstrom scale. The researchers’ technique enables the study of whole and intact cells over individual proteins — all the way down to the distance between two adjacent bases in DNA. The researchers, from the group of Ralf Jungmann, called the technique resolution enhancement by sequential imaging, or RESI. Current SMLM resolve structures on the order of 10 nm by temporally separating the structures’ individual fluorescence emission. As individual targets stochastically light up in an otherwise dark field of view, their location can be determined with subdiffraction precision. The SMLM technique of DNA points accumulation for imaging in nanoscale topography, or DNA-PAINT, uses the transient hybridization of dye-labeled DNA “imager” strands to their target-bound complements to achieve the light-up necessary to achieve superresolution. To date, however, neither DNA-PAINT nor other superresolution methods have been able to resolve the smallest cellular structures. RESI builds on DNA-PAINT and capitalizes on its ability to encode target identity via DNA sequences. By labeling adjacent targets, too close to each other to be resolved even by superresolution microscopy, with different DNA strands, an additional degree of differentiation — a barcode — is introduced into the sample. By sequentially imaging first one and then the other sequence to thereby capture the full target, the strands can now be unambiguously separated. Critically, as they are imaged sequentially, the targets can be arbitrarily close to one other, which is a dynamic that existing techniques are unable to resolve. Further, RESI does not require specialized instrumentation and can be applied using any standard fluorescence microscope. RESI enables microscopy across length scales at angstrom resolution: from whole cells over individual proteins down to the distance between two adjacent bases in DNA. Courtesy of Max Iglesias/Max Planck Institute of Biochemistry. To demonstrate RESI’s leap in resolution compared to other methods, the researchers sought to resolve the separation between individual bases along a double helix of DNA, which is separated by less than 1 nm. They designed a DNA origami nanostructure that presented single-stranded DNA sequences that protrude from a double helix at one base pair distance. The researchers then imaged these single strands sequentially and resolved a distance of 0.85 nm, or 8.5 Å between adjacent bases. They accomplished these measurements with a precision of 1 Å, or one ten-billionth of a meter. According to the researchers, the technique is universal, with application beyond DNA nanostructures. In separate tests, Jungmann and his team investigated the molecular mode of action of rituximab, an anti-CD20 monoclonal antibody used for treatment of CD20-positive blood cancer. Investigating the effects of such drug molecules on molecular receptor patterns has been beyond the spatial resolution capabilities of traditional microscopy techniques. Understanding whether and how such patterns change in health and disease as well as upon treatment is important for mechanistic research and the design of targeted therapies. The researchers used RESI to reveal the natural arrangement of CD20 receptors in untreated cells as dimers and uncovered how CD20 re-arranged to chains of dimers upon drug treatment. Because RESI is performed in whole, intact cells, the technique closes the gap between purely structural techniques such as x-ray crystallography or cryogenic electron microscopy and traditional lower-resolution whole-cell imaging approaches. The research was published in Nature (www.doi.org/10.1038/s41586-023-05925-9).