The project has made a significant breakthrough in imaging individual molecules inside living cells using a technology called ZMW. In the past, it was very challenging to observe single molecules using fluorescence techniques because cells contain a lot of proteins, making it difficult to distinguish individual molecules. However, the project has found a promising solution by using ZMW, which are tiny holes smaller than the wavelength of light, in a thin metal film. These holes create extremely small spaces where molecules can be observed. In collaboration with a specialized research group, the project has developed a new ZMW technology specifically designed for imaging molecules in live cells. This technology has opened up new possibilities for studying cells in greater detail. One exciting discovery is that human cells can form stable extensions into these tiny holes, allowing us to observe fluorescently labeled proteins at the level of individual molecules over long periods of time. With this new approach, we can observe single molecules with exceptional clarity and accuracy, even when there is a lot of background noise from other molecules in the cell. Traditional microscopy techniques like confocal and TIRF have limitations in dealing with high levels of background noise, but our method overcomes these challenges. It can be easily implemented on standard fluorescence microscopes, making it accessible for many researchers working with different biological systems.
Additionally, we studied a wide range of sizes of the ZMWs to provide selection guidelines for optimal optical properties and cell compatibility. This comprehensive analysis gives researchers valuable insights for designing experiments using ZMW technology. In summary, this project has developed an advanced technique using ZMW technology to visualize individual molecules inside living cells. This breakthrough allows us to study cells in ways that were previously not possible, opening up new avenues of research in the field of cell biology.
The results of this MSCA will be reported in:
(1) A peer-reviewed journal as a research paper on live-cell single-molecule imaging method using zero-mode waveguide
(2) A peer-reviewed journal as a research paper on translation elongation heterogeneity
(3) The 12th Single Molecule Localization Microscopy Symposium in 2023
For research training and transfer of knowledge, the fellow attended 4 conferences (2021 EMBL conference: Protein Synthesis and Translational control, 2022 NWO Biophysics, 2022 EMBL symposium: The complex life of RNA, 2022 RNA – Beyond its Genetic Code)
