Angstrom resolution fluorescence microscopy

May 25, 2023

(Nanowerk News) The cell, the basic unit of life, contains a myriad of intricate structures, processes and mechanisms that support and sustain living systems. Many components of the cellular core, such as DNA, RNA, proteins, and lipids, are only a few nanometers in size. This makes them much smaller than the resolution limits of traditional light microscopes. The exact composition and arrangement of these molecules and structures is often unknown, resulting in a lack of mechanistic understanding of the basic aspects of biology.

In recent years, super-resolution techniques have made leaps and bounds to resolve many sub-cellular structures below the classical light diffraction limit. Single molecule localization microscopy, or SMLM, is a super-resolution approach that can resolve structures on the order of ten nanometers in size by temporarily separating the respective fluorescence emissions. When individual targets flash stochastically (blink) in a dark field of view, their location can be determined with sub-diffraction precision. DNA-PAINT, discovered by Jungmann’s group, is an SMLM technique that uses temporary hybridization of dye-labeled DNA “imager” strands to target-bound complements to achieve the flicker required for super-resolution. However, to date, even DNA-PAINT has not been able to resolve the smallest cellular structures. Improved Resolution with Sequential Imaging allows microscopy to traverse the length scale at Ångström resolution: From the entire cell over individual proteins to the distance between two adjacent bases in DNA. (Image: Max Iglesias, MPI from Biochemistry)

In the current study (Natural, “Angström resolution fluorescence microscopy”) led by first co-authors Susanne Reinhardt, Luciano Masullo, Isabelle Baudrexel and Philipp Steen together with Jungmann, the team introduces a new approach in super-resolution microscopy that enables fundamentally “infinite” spatial resolution. The new technique, called “Resolution Enhancement by Sequential Imaging”, or RESI for short, takes advantage of DNA-PAINT’s ability to encode a target’s identity through a unique DNA sequence.

By labeling targets that are close together, too close to each other to be resolved even by super-resolution microscopy, with different DNA strands, additional levels of differentiation (bar codes) are introduced into the sample. By imaging one sequence first, and then another sequence (and thus targeting), they can now be clearly separated. Critically, because they are imaged sequentially, targets can be arbitrarily close to one another, something that other techniques cannot resolve.

Additionally, RESI does not require special instrumentation, in fact it can be applied using any standard fluorescence microscope, making it easily accessible to almost any researcher.

To demonstrate RESI’s leap in resolution, the team set a challenge to solve one of the smallest spatial distances in a biological system: Separations between individual bases along the DNA double helix, spaced less than a nanometer (a billionth of a meter). separated. By designing the DNA origami nanostructure in such a way as to present single-stranded DNA sequences protruding from the double helix at a distance of one base pair and then imaging these single strands sequentially, the research team resolved the 0.85 nm (or 8.5 Ångström) distance between the nearby. base, previously unimaginable feat. The researchers completed these measurements with a precision of 1 Ångström, or one ten billionth of a meter, underscoring the unprecedented capabilities of the RESI approach.

Importantly, this technique is universal and not limited to applications in DNA nanostructures. To this end, the team investigated the molecular mode of action of Rituximab, an anti-CD20 monoclonal antibody first approved in 1997 for the treatment of CD20-positive blood cancer. However, investigating the effects of such drug molecules on molecular receptor patterns has exceeded the spatial resolution capabilities of traditional microscopy techniques.

Understanding whether and how such patterns change in health and disease and in medicine is important not only for basic mechanistic research, but also for designing new disease targeted therapies. Using RESI, Jungmann and his team were able to unravel the natural arrangement of the CD20 receptor in cells that were not treated as dimers and uncover how CD20 rearranges into dimer chains upon drug treatment. Insights at the single protein level now help elucidate Rituximab’s mode of molecular action.

Because RESI is performed in whole, intact cells, this technique closes the gap between purely structural techniques such as X-ray crystallography or cryogenic electron microscopy and traditional low-resolution whole-cell imaging approaches. Jungmann and his team believe that “this unprecedented technique is a true game changer not only for super-resolution, but for biological research as a whole.”

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