This makes them considerably smaller than the resolution limitation of conventional light microscopy. The specific composition and arrangement of these molecules and structures are thus frequently unidentified, resulting in a lack of mechanistic understanding of basic aspects of biology.
Recently, super-resolution strategies have actually made leaps and bounds to solve numerous sub-cellular structures below the classical diffraction limitation of light.
Single-molecule localization microscopy, or SMLM, is a super-resolution technique that can solve structures on the order of ten nanometers in size by temporally separating their individual fluorescence emission. As specific targets stochastically illuminate (they blink) in an otherwise dark field of vision, their location can be identified with sub-diffraction precision.
DNA-PAINT, invented by the Jungmann group, is an SMLM technique that utilizes short-term hybridization of dye-labeled DNA “imager” hairs to their target-bound matches to attain the required blinking for super-resolution. To date, even DNA-PAINT has not been able to solve the tiniest cellular structures.
Unlimited resolution
In the current study led by co-first authors Susanne Reinhardt, Luciano Masullo, Isabelle Baudrexel, and Philipp Steen together with Jungmann, the team introduces a novel method in super-resolution microscopy that allows basically “unlimited” spatial resolution.
The new method, called “Resolution Enhancement by Sequential Imaging”, or RESI for short, capitalizes on the ability of DNA-PAINT to encode target identity by means of special DNA series. By identifying nearby targets, too close to each other to be dealt with even by super-resolution microscopy, with different DNA strands, an extra degree of distinction (a barcode) is introduced into the sample.
By sequentially imaging the first one, and then the other series (and consequently target), they can now be unambiguously separated. Seriously, as they are imaged sequentially, the targets can be arbitrarily close to each other, something no other technique can fix. Furthermore, RESI does not need specific instrumentation, in fact, it can be applied utilizing any basic fluorescence microscopic lense, making it quickly available for practically all scientists.
To show RESIs leap in resolution, the group set themselves the challenge of solving one of the smallest spatial distances in a biological system: The separation between private bases along a double helix of DNA, spaced less than one nanometer (a billionth of a meter) apart.
By designing a DNA origami nanostructure such that it presents single-stranded DNA series that extend from a double helix at one base set distance and after that imaging these single hairs sequentially, the research study team resolved a distance of 0.85 nm (or 8.5 Ångström) between surrounding bases, a previously inconceivable accomplishment.
The scientists accomplished these measurements with a precision of 1 Ångström, or one ten-billionth of a meter, highlighting the extraordinary abilities of the RESI technique.
Importantly, the method is universal and not restricted to applications in DNA nanostructures. To this end, the group examined the molecular mode of action of Rituximab, an anti-CD20 monoclonal antibody that was first authorized in 1997 for the treatment of CD20-positive blood cancer.
Investigating the effects of such drug particles on molecular receptor patterns has actually been beyond the spatial resolution capabilities of standard microscopy strategies. Comprehending whether and how such patterns change in health and disease along with upon treatment is not only essential for basic mechanistic research however also for developing novel targeted illness therapies.
Utilizing RESI, Jungmann and his group had the ability to reveal the natural arrangement of CD20 receptors in neglected cells as dimers and reveal how CD20 re-arranged to chains of dimers upon drug treatment. The insights on the single-protein level now assist to clarify the molecular mode of action of Rituximab.
As RESI is carried out in whole, undamaged cells, the method closes the gap in between purely structural techniques such as X-ray crystallography or cryogenic electron microscopy and traditional lower-resolution whole-cell imaging methods.
Jungmann and his team are convinced that “this unprecedented technique is a real game-changer not just for super-resolution, but for biological research study as a whole.”
Recommendation: “Ångström-resolution fluorescence microscopy” by Susanne C. M. Reinhardt, Luciano A. Masullo, Isabelle Baudrexel, Philipp R. Steen, Rafal Kowalewski, Alexandra S. Eklund, Sebastian Strauss, Eduard M. Unterauer, Thomas Schlichthaerle, Maximilian T. Strauss, Christian Klein and Ralf Jungmann, 24 May 2023, Nature.DOI: 10.1038/ s41586-023-05925-9.
RESI enables microscopy throughout length scales at Ångström resolution: From entire cells over individual proteins down to the range between 2 adjacent bases in DNA. Credit: MPI of Biochemistry/ Max Iglesias
Researchers have actually attained Ångström-level resolution utilizing DNA-tagged fluorescent microscopy.
Ralf Jungmanns research group at limit Planck Institute of Biochemistry and the Ludwig Maximilian University of Munich have actually achieved a significant advance in fluorescence microscopy.
They have actually established an innovative technique called Resolution Enhancement by Sequential Imaging, which incredibly improves the resolution of fluorescence microscopy to an Ångström scale. This unique method is set to revolutionize our exploration of biological systems by offering hitherto extraordinary detail.
Cells, the essential systems of life, include a variety of intricate structures, procedures, and systems that promote and perpetuate living systems. Numerous cellular core parts, such as DNA, RNA, proteins, and lipids, are simply a few nanometers in size.