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Fluorescence-based 3D targeting of FIB-SEM acquisition of small volumes in large samples

By Paolo Ronchi, Pedro Machado, Edoardo D'Imprima, Giulia Mizzon, Benedikt Thomas Best, Lucia Cassella, Sebastian Schnorrenberg, Marta Garcia Montero, Martin Jechlinger, Anne Ephrussi, Maria Leptin, Julia Mahamid, Yannick Schwab

Posted 19 Jan 2021
bioRxiv DOI: 10.1101/2021.01.18.427072

Cells are three dimensional objects. Therefore, 3D electron microscopy is often crucial for correct interpretation of ultrastructural data. Today samples are frequently imaged in 3D at ultrastructural resolution using volume Scanning Electron Microscopy (SEM) methods such as Focused Ion Beam (FIB) SEM and Serial Block face SEM. While these imaging modalities allow for automated data acquisition, precise targeting of (small) volumes of interest within a large sample remains challenging. Here, we provide an easy and reliable approach to target FIB-SEM acquisition of fluorescently labelled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeting based on confocal acquisition of the fluorescence signal in the resin block. Targeted trimming of the block exposes the cell of interest and laser branding of the surface after trimming creates landmarks to precisely position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as a 3D culture of mouse primary mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.

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