Data underlying the publication: Thickness and quality controlled fabrication of fluorescence-targeted frozen-hydrated lamellae

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<br>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 interference for fine-grained feedback on thickness uniformity below 500 nm. Finally, we present a fluorescence-targeted, quality-controlled workflow for frozen-hydrated lamellae, benchmarked with excellent agreement to EFTEM measurements and tomograms from electron cryo-tomography.<br>General descriptionAll data underlying the manuscript figures is stored in this repository. Experiments have been carried out at the Department of Imaging Physics and Kavli Institute of Nanoscience at Delft University of Technology (Delft, NL) in 2023 and 2024.<br>The repository contains images acquired in a transmission electron microscope, scanning electron microscope, focused ion beam, fluorescence microscope and a reflection light microscope.<br>