The latest super-resolution microscopy techniques currently approach optical resolutions of a few nanometers. This corresponds to a resolution in the region of biological molecular sizes. However, because there were no biomolecular reference systems that could be labeled with dyes at precisely specified places at a few nanometers, it was not possible to verify the resolution reached on cellular building blocks such as multiprotein complexes.
A turning point has now been offered by a team led by Professor Markus Sauer and Dr. Gerti Beliu from the Rudolf Virchow Centre - Center for Integrative and Translational Bioimaging at Julius-Maximilians-Universität (JMU) Würzburg in Bavaria, Germany. They describe the PicoRulers (Protein-based Imaging Calibration Optical Rulers), innovative biocompatible molecular rulers, in the journal Advanced Materials.
Through the use of click chemistry and genetic code expansion, the team has successfully built these bespoke molecular rulers. Fluorescence microscopy may make use of them as accurate biomolecular reference structures.
Technological Masterpiece: Precision at Molecular Level
The three-part protein PCNA (Proliferating Cell Nuclear Antigen), which is essential for DNA replication and repair, serves as the foundation for the PicoRulers. This protein has been altered such that fluorescent dyes or other molecules may be selectively clicked onto it with the least amount of linkage error by carefully introducing non-natural amino acids at precisely determined places.
This allows researchers to evaluate the resolution of the latest methods for super-resolution microscopy on a precisely defined cellular biomolecule with previously unheard-of accuracy.
The ability to resolve real biological structures at sub-10-nanometre level marks a new era in biological imaging. Compared to previously used artificial macromolecules, our PicoRulers are not only characterized by their biological compatibility. They also enable unrivaled precision for testing resolution under realistic conditions.
Markus Sauer, Professor, Rudolf Virchow Centre-Center for Integrative and Translational Bioimaging, Julius-Maximilians-Universität
Opening the Door to Investigate Complex Processes in Cells
This method has far-reaching applications outside of the conventional confines of microscopy.
Our PicoRulers are not only a tool for more precise measurements, but also open the door to a deeper and more detailed investigation of complex processes that take place within our cells.
Dr. Gerti Beliu, Research Group Leader, Rudolf Virchow Center
Strong Potential for Future Applications
Long-term changes in biological and medical imaging with molecular resolution might result from the further development of PicoRulers. They enable the first-ever validation and enhancement of new super-resolution microscopy techniques’ resolution potential on biological material. As a result, they will be a useful tool in the quest to understand the molecular structure and interactions of biomolecules in cells.
Helmerich, D. A., et al. (2023). Pcna as Protein-Based Nanoruler for Sub-10 Nm Fluorescence Imaging. Advanced Materials. doi:10.1002/adma.202310104.