IntroductionData underlying the publication: Thickness and quality controlled fabrication of fluorescence-targeted frozen-hydrated lamellae.Publication DOI: https://doi.org/10.1016/j.crmeth.2025.101004Authors: D.B. Boltje, R. Skoupy, C. Taisne, W. H. Evers, A.J. Jakobi, J.P. HoogenboomRepository created by Daan Boltje
AbstractCryogenic FIB milling is essential for fabricating thin lamella-shaped samples out of frozen-hydrated cells for high-resolution structure determination. Structural information can only be resolved at high resolution if the lamella thickness is between 100 to 200 nm. While the lamella fabrication workflow has improved significantly since its conception, quantitative, live feedback on lamella thickness, quality, and biological target inclusion remains lacking. Using coincident light microscopy integrated into the FIB-SEM, we present three strategies that enable accurate, live control during lamella fabrication. First, we combine 4D-STEM with FM targeting to determine lamella thickness. Second, with RLM, we screen target sites for ice contamination and monitor lamella thickness and protective Pt coating integrity during FIB milling. Third, we exploit thin-film