High-precision targeting workflow for volume electron microscopy.
Animals
Drosophila melanogaster
/ metabolism
Epithelial Cells
/ metabolism
Female
Gene Expression
Genes, Reporter
Granulosa Cells
/ metabolism
Green Fluorescent Proteins
/ genetics
HeLa Cells
Humans
Larva
/ metabolism
Luminescent Proteins
/ genetics
Mammary Glands, Animal
/ metabolism
Mice
Microscopy, Electron, Scanning
/ instrumentation
Organoids
/ metabolism
Single-Cell Analysis
/ instrumentation
Staining and Labeling
/ methods
Theca Cells
/ metabolism
Trachea
/ metabolism
Workflow
Red Fluorescent Protein
Journal
The Journal of cell biology
ISSN: 1540-8140
Titre abrégé: J Cell Biol
Pays: United States
ID NLM: 0375356
Informations de publication
Date de publication:
06 09 2021
06 09 2021
Historique:
received:
16
04
2021
revised:
27
05
2021
accepted:
06
06
2021
entrez:
23
6
2021
pubmed:
24
6
2021
medline:
10
11
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 34160561
pii: 212433
doi: 10.1083/jcb.202104069
pmc: PMC8225610
pii:
doi:
Substances chimiques
Luminescent Proteins
0
enhanced green fluorescent protein
0
Green Fluorescent Proteins
147336-22-9
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM084947
Pays : United States
Informations de copyright
© 2021 Ronchi et al.
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