Chowdhury Rajdeep, Mimoso Tiago, Chouaib Abed Alrahman, Mougios Nikolaos, Krah Donatus, Opazo Felipe, Köster Sarah, Rizzoli Silvio O, Shaib Ali H
Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany.
Department of Chemistry, GITAM School of Science, GITAM, Hyderabad, Telangana, India.
Commun Biol. 2025 Mar 26;8(1):499. doi: 10.1038/s42003-025-07967-3.
Expansion microscopy (ExM) is continually improving, and new ExM variants need to be validated on well-defined biological structures. There is no consensus on validation structures for ExM, especially as nuclear pore complexes or DNA nanorulers are not popular for ExM studies. Here we propose that microtubules should be used for ExM validation. The diameter of microtubules immunostained using primary and secondary antibodies is sufficiently large for the validation of techniques with resolutions better than 50 nm. For techniques with higher precision (up to ~10 nm), microtubules can be assembled and imaged in vitro, using a protocol that we introduce here. Alternatively, a cellular extraction procedure can be employed, followed by labeling the peptide chains of the tubulin molecules with NHS-ester fluorophores. Finally, for nanometer-scale techniques, single tubulin molecules can be analyzed. We conclude that microtubules are valuable structures for the validation of ExM and related technologies.
扩展显微镜技术（ExM）在不断改进，新的ExM变体需要在明确的生物结构上进行验证。对于ExM的验证结构尚无共识，尤其是核孔复合体或DNA纳米尺在ExM研究中并不常用。在此，我们建议将微管用于ExM验证。使用一抗和二抗免疫染色的微管直径足够大，可用于分辨率优于50nm的技术验证。对于精度更高（高达约10nm）的技术，微管可以在体外组装并成像，我们在此介绍了相关方案。或者，可以采用细胞提取程序，然后用NHS-酯荧光团标记微管蛋白分子的肽链。最后，对于纳米级技术，可以分析单个微管蛋白分子。我们得出结论，微管是用于验证ExM及相关技术的有价值结构。
