PT - JOURNAL ARTICLE AU - Boltje, Daan B. AU - Skoupy, Radim AU - Taisne, Clémence AU - Evers, Wiel H. AU - Jakobi, Arjen J. AU - Hoogenboom, Jacob P. TI - Thickness and quality controlled fabrication of fluorescence-targeted frozen-hydrated lamellae AID - 10.1101/2024.07.04.602102 DP - 2024 Jan 01 TA - bioRxiv PG - 2024.07.04.602102 4099 - http://biorxiv.org/content/early/2024/07/08/2024.07.04.602102.short 4100 - http://biorxiv.org/content/early/2024/07/08/2024.07.04.602102.full AB - Cryogenic focused ion beam (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 and 200 nm. While the lamella fabrication workflow has undergone significant improvements since its conception, quantitative, live feedback on lamella thickness and quality is still lacking. Taking advantage of a coincident light microscopy integrated into the FIB-SEM, we present three different strategies that together allow accurate, live control during lamella fabrication. First, we combine 4D-STEM with fluorescence microscope (FM) targeting to determine the lamella thickness. Second, with reflected light microscopy (RLM) we screen target sites for ice contamination and monitor lamella thickness and integrity of the protective Pt coating during FIB milling. Third, we exploit thin-film interference to obtain fine-grained feedback on thickness uniformity below 500 nm. We finally present a full workflow for fluorescence-targeted and quality controlled fabrication of frozen-hydrated lamellae, benchmarked with excellent agreement to energy filtered transmision electron microscopy (EFTEM) measurements and reconstructed tomograms obtained with electron cryo-tomography.Competing Interest StatementCT and WHE declare no competing interest. Parts of this work are covered in; NL Patent 2032641 (RS, JPH, AJJ, DBB) and 2028497 (DBB). DBB is an employee of Delmic BV. JPH has a financial interest in Delmic BV.