BIO407 Group 5: Segmentation of mitochondria and nuclei from mouse cerebellum imaged by FIB/SEM

# REMBI metadata ## Study:This data is part of the practical course BIO407 2026 at the University of Zurich, titled 'Advanced Microscopy: From preparation to data and visualization'. ## Group:Andreas Enderli, ORCID id: 0009-0003-8184-0035Pia Hartvigsen, ORCID id: 0009-0008-2679-8731Alessandra Rossetti, ORCID id: 0009-0002-2169-119X ## Study Component:Job: Electron microscopyTask: "Segmentation of mitochondria and nuclei from mouse cerebellum imaged by FIB/SEM". ## Biosample:Cerebellar mouse tissue used exclusively for teaching purposes originated from surplus material initially collected for the study "Maheshwari U, Mateos JM, Weber-Stadlbauer U, Ni R, Tamatey V, Sridhar S, Restrepo A, de Jong PA, Huang SF, Schaffenrath J, Stifter SA, Szeri F, Greter M, Koek HL, Keller A. Inorganic phosphate exporter heterozygosity in mice leads to brain vascular calcification, microangiopathy, and microgliosis. Brain Pathol. 2023 Nov;33(6):e13189. doi: 10.1111/bpa.13189. Epub 2023 Jul 28. PMID: 37505935; PMCID: PMC10580014." No additional animals were used or sacrificed for teaching. ## Specimen:200um vibratome section from cerebellum mouse tissue. Mice were transcardially perfused under anesthesia (ketamine–xylazine cocktail) with ice-cold PBS followed by freshly made Karnovsky's fixative—2% PFA (Polysciences, no. 18814-10), 2.5% glutaraldehyde (Polysciences, no. 01909-10) in 0.1 M cacodylate buffer (pH 7.4) (Polyscience, no. 18661-500). Brains were sectioned with a vibratome (VT1200S, Leica) at 200um thickness. Sections were kept in Karnovsky's fixative until sample preparation for EM. Cerebellar sections were washed in 0.1 M cacodylate buffer before incubation with 1.5% potassium ferrocyanide and 2% OsO4 in 0.1 M cacodylate buffer for 1 h on ice, followed by 0.1 % TCH (thiocarbohydrazide) 1 hour, 2% OsO4 1 hour and 1% aqueous uranyl acetate overnight at 4°C. Samples were further contrasted with Walton's lead aspartate at 50°C and dehydrated in an ethanol series, embedded in Epon/Araldite (Sigma-Aldrich) and polymerized at 60°C for 28 hours. Resin blocks were mounted on a SEM stub with conductive carbon and 5 nm carbon coated was deposited on the surface of the block (CCU-010; Safematic). ## Image Acquisition:Electron microscopyImaging instrument: FIB/SEM Auriga 40 CrossBeam. SEM 1.8 kV acceleration voltage, dwell time: 5us, ESB detector, detector grid: 400V. FIB aperture 600pA at 30kV. Automatic image acquisition with Zeiss Atlas 5. Image dimensions:Pixel size: 10 nm. 3D stack from sample 10150-ec-02-sa-01-FIB-RUN-02 with crop region selected by students from the large FIB/SEM data. ## Image Data:260304_FIB_SEM_aligned_nucleus_AR_BIO407.tif260304_FIB_SEM_croped_nucleus_AR_407.tif260304_FIB_SEM_rawdata_AR_407.tif260304_FIB_SEM_segmentation_nucleus_AR_407.tif260304_FIB-RUN-02_rawdat_unaligned_AR_407.tif260305_FIB_SEM_mobieproject_nucleus_mitochondria_AR_407.tif260305_FIB_SEM_segmented_mitochondria_ARN_407.tif