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High-precision targeting workflow for volume electron microscopy.
- Source :
-
The Journal of cell biology [J Cell Biol] 2021 Sep 06; Vol. 220 (9). Date of Electronic Publication: 2021 Jun 23. - Publication Year :
- 2021
-
Abstract
- 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.<br /> (© 2021 Ronchi et al.)
- Subjects :
- Animals
Drosophila melanogaster metabolism
Epithelial Cells metabolism
Epithelial Cells ultrastructure
Female
Gene Expression
Genes, Reporter
Granulosa Cells metabolism
Green Fluorescent Proteins genetics
Green Fluorescent Proteins metabolism
HeLa Cells
Humans
Larva metabolism
Larva ultrastructure
Luminescent Proteins genetics
Luminescent Proteins metabolism
Mammary Glands, Animal metabolism
Mice
Microscopy, Electron, Scanning instrumentation
Organoids metabolism
Organoids ultrastructure
Single-Cell Analysis instrumentation
Single-Cell Analysis methods
Theca Cells metabolism
Trachea metabolism
Workflow
Red Fluorescent Protein
Drosophila melanogaster ultrastructure
Granulosa Cells ultrastructure
Mammary Glands, Animal ultrastructure
Microscopy, Electron, Scanning methods
Staining and Labeling methods
Theca Cells ultrastructure
Trachea ultrastructure
Subjects
Details
- Language :
- English
- ISSN :
- 1540-8140
- Volume :
- 220
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- The Journal of cell biology
- Publication Type :
- Academic Journal
- Accession number :
- 34160561
- Full Text :
- https://doi.org/10.1083/jcb.202104069