Researchers at the University of Würzburg, Germany, have developed protein-based molecular rulers that, the team says, make it possible to test the optical resolution of the latest super-resolution microscopy methods on biomolecules in the sub-10-nm range under realistic conditions. [Image: Gerti Beliu, DALL-E 3 / University of Würzburg]
More than two decades after breaking the diffraction limit, the field of super-resolution microscopy continues to give biologists an unprecedented look inside cells with the use of light. Since its advent, spatial resolution of the Nobel Prize–winning technology has improved from tens of nanometers to the one-digit nanometer range.
Now, researchers in Germany have reportedly developed a novel reference structure, known as PicoRulers (Protein-based Imaging Calibration Optical Rulers), to more accurately estimate the spatial resolution of super-resolution microscopy methods (Adv. Mater., doi: 10.1002/adma.202310104). The team believes that its PicoRulers, based on multi-labeled oligomeric proteins, have several advantages over previous reference structures, including biocompatibility, stability and modularity.
A biologically relevant ruler
The creation of PicoRulers originated from a desire by study authors Markus Sauer and Gerti Beliu, University of Würzburg, to further enhance the resolution capabilities of super-resolution fluorescence microscopy, especially in living cells. In his research, Sauer applies super-resolution fluorescence imaging to questions in neurobiology and immunology, while Beliu’s lab aims to utilize novel biochemical compounds for stoichiometric fluorescence labeling without altering biological function.
Sauer and Beliu argue that evaluation for cellular imaging applications should use biologically relevant reference structures or rulers.
Currently, the resolving power of newer super-resolution microscopy methods is demonstrated with DNA origami structures, which allow for the attachment of fluorophores at nanometer distances. However, Sauer and Beliu argue that evaluation for cellular imaging applications should use biologically relevant reference structures or rulers.
“Our team aimed to create biocompatible, precise molecular rulers that could calibrate the latest super-resolution microscopy techniques with nanometer-level accuracy, thus pushing the boundaries of imaging resolution in the realm of cellular molecules,” said Sauer.
Calibration tool for researchers
The researchers designed a reference system based on the protein, Proliferating Cell Nuclear Antigen (PCNA), which plays a pivotal role in DNA replication and repair. PCNA is comprised of three identical units of polypeptide, with inner and outer diameters of 3.4 nm and 8.0 nm, respectively.
Through genetic code expansion and click chemistry, PicoRulers incorporate non-canonical amino acids (ncAAs)—as in, ones that are not naturally genetically encoded—at specific positions in the PCNA protein. This modification enables the site-specific attachment of organic dyes with minimal linkage error. The researchers inserted three ncAAs into the PCNA labeled at well-defined 6 nm distances and labeled them with tetrazine-dyes and tetrazine-functionalized oligonucleotides.
Overall, they hope that the development of PicoRulers will offer a calibration tool to verify and enhance the accuracy of newer techniques.
Lastly, Sauer, Beliu and their colleagues employed the super-resolution microscopy method known as DNA-PAINT to successfully resolve the regular triangle structure of the 6 nm PicoRuler. Overall, they hope that the development of PicoRulers will offer a calibration tool to verify and enhance the accuracy of newer techniques.
“Our team focuses now on expanding the range of biomolecules that can be used as PicoRulers, exploring different proteins or complexes that offer diverse applications in biological imaging,” said Beliu. “Another direction could be the exploration of delivering PicoRulers into cells, possibly through methods like microinjection or functionalization with cell-penetrating peptides, to conduct intracellular super-resolution imaging experiments.”