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>

数据集关联文献简介：针对荧光靶向冷冻水合薄片的厚度与质量可控制备方法。文献DOI：https://doi.org/10.1016/j.crmeth.2025.101004 作者：D.B. Boltje、R. Skoupy、C. Taisne、W. H. Evers、A.J. Jakobi、J.P. Hoogenboom 本数据集仓库由Daan Boltje创建

摘要：冷冻聚焦离子束铣削（Cryogenic FIB milling）是从冷冻水合细胞中制备超薄薄片状样品以开展高分辨结构解析的核心技术。仅当薄片厚度处于100~200 nm区间时，方可获得高分辨率的结构信息。尽管薄片制备流程自问世以来已有显著改进，但目前仍缺乏针对薄片厚度、质量以及生物靶标保留情况的定量实时反馈方案。本研究借助集成于聚焦离子束-扫描电子显微镜（FIB-SEM）中的同步光学显微镜技术，提出了三种可在薄片制备过程中实现精准实时调控的策略：其一，将四维扫描透射电子显微镜（4D-STEM）与荧光靶向（Fluorescence Microscopy, FM）技术结合以测定薄片厚度；其二，利用反射光显微镜（Reflection Light Microscopy, RLM）筛选无冰污染的靶位点，并在FIB铣削过程中实时监测薄片厚度与保护性铂（Pt）涂层的完整性；其三，借助薄膜干涉效应实现500 nm以下厚度均匀性的精细化反馈。最终，我们构建了一套荧光靶向且经过质量管控的冷冻水合薄片制备流程，经验证，该流程的测量结果与能量过滤透射电子显微镜（Energy-Filtered Transmission Electron Microscopy, EFTEM）及冷冻电子断层扫描的断层图像结果高度吻合。

数据集概况：本仓库存储了支撑该手稿所有图表的全部实验数据。相关实验于2023至2024年间在代尔夫特理工大学成像物理系与卡夫利纳米科学研究所（荷兰代尔夫特）完成。本仓库包含透射电子显微镜、扫描电子显微镜、聚焦离子束、荧光显微镜及反射光显微镜采集的图像数据。