Dr. Gerti Beliu
University of Würzburg

Super-resolution microscopy (SRM) has revolutionized our ability to visualize cellular structures and processes at molecular resolution, yet achieving this level of detail depends on precise and efficient fluorescent labeling techniques. A major question driving our research is how to develop labeling strategies that maximize localization precision and accuracy while minimizing interference with cellular function. Addressing this is essential to meet the increasing demands of SRM, particularly as we approach sub-10 nm resolutions where label size and linkage error can critically impact accuracy.[1]

To overcome these challenges, we employ genetic code expansion (GCE) to incorporate unnatural amino acids (uAAs) into proteins of interest, followed by bioorthogonal click chemistry for the selective attachment of small, photophysically optimized dyes.[2] This approach allows for site-specific labeling with minimal linkage error, a crucial factor for achieving molecular-level accuracy. Specifically, we utilize strained alkenes such as trans-cyclooct-2-ene (TCO) that react with tetrazine-functionalized dyes in an ultrafast and bioorthogonal manner, enabling quantitative live-cell labeling without wash steps. Our results demonstrate that this strategy enhances spatial resolution and improves signal-to-noise ratios, supporting high-quality imaging in live-cell environments.

Our findings suggest that optimized uAA-based labeling can drive SRM capabilities further by reducing background fluorescence and maintaining biological function, as shown in applications such as protein-protein interaction assays and virus-host interaction studies. Our current work focuses on extending these labeling techniques for multicolour SRM and developing even smaller, more efficient fluorophores. These advancements are currently expanding the potential of SRM in cellular and molecular biology, allowing for deeper insights into complex biological systems in neuroscience, structural biology and virus imaging.[3-7]

References:

[1] Lelek, M., Gyparaki, M.T., Beliu, G. et al. Single-molecule localization microscopy. Nat Rev  Methods Primers 1, 39 (2021), doi:10.1038/s43586-021-00038-x

[2] Beliu, G. et al. Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy. Commun Biol 2, 261 (2019), doi:10.1038/s42003-019-0518-z  

[3] Neubert*, F., Beliu*. G., et al. Bioorthogonal click chemistry enables site-specific fluorescence labeling of functional NMDA receptors for super-resolution imaging. Angew. Chem. Int. Ed. 57, 16364-16369 (2018), doi:/10.1002/anie.201808951  

[4] Beliu G. et al. Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain. Mol. Cell. 81: 905-921 (2021), doi:10.1016/j.molcel.2020.12.042  

[5] Bessa-Neto*, D., Beliu*, G. Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids allows access to masked epitopes in live neurons. Nature Communications (2021), doi:10.1038/s41467-021-27025-w

[6] Helmerich, D.A., Budiarta, M., Taban, D., Doose, S., Beliu, G.*, Sauer, M.* PCNA as a protein-based nanoruler for sub-10 nm fluorescence imaging. Adv. Mater. (2024), doi:10.1002/adma.202310104      

[7] Jungblut, M., Backes, S., Streit, M., Gasteiger, G., Doose, S., Sauer, M., Beliu, G. Re-engineered pseudoviruses for precise and robust 3D mapping of viral infection. ACS Nano 17, 21822-21828 (2023), doi:10.1021/ acsnano.3c07767