期刊
JOURNAL OF CELL BIOLOGY
卷 220, 期 9, 页码 -出版社
ROCKEFELLER UNIV PRESS
DOI: 10.1083/jcb.202104069
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资金
- Transgenic RNAi Project at Harvard Medical School (National Institutes of Health/National Institute of General Medical Sciences) [R01-GM084947]
- European Molecular Biology Laboratory
- Deutsche Forschungsgemeinschaft (Germany) [DFG-FOR 2333, EP 37/2-1, EP 37/4-1]
- European Molecular Biology Laboratory interdisciplinary postdoctoral program under Marie SklodowskaCurie Actions COFUND (EI4POD)
- European Research Council [760067]
- Electron Microscopy Core Facility
- Advanced Light Microscopy Facility
- animal facility at European Molecular Biology Laboratory
- European Research Council (ERC) [760067] Funding Source: European Research Council (ERC)
Cells are 3D objects, and volume EM is essential for interpreting ultrastructural data accurately. FIB-SEM methods such as focused ion beam are now commonly used, but precise targeting of volumes of interest within large samples remains challenging. This study introduces a workflow for targeting specific cells or subcellular structures with micrometer precision using fluorescence preservation and confocal maps to guide FIB-SEM acquisition, allowing for the discovery of new ultrastructural details.
Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)-SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse 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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