High-precision targeting workflow for volume electron microscopy

J Cell Biol. 2021 Sep 6;220(9):e202104069. doi: 10.1083/jcb.202104069. Epub 2021 Jun 23.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Drosophila melanogaster / metabolism
  • Drosophila melanogaster / ultrastructure*
  • Epithelial Cells / metabolism
  • Epithelial Cells / ultrastructure
  • Female
  • Gene Expression
  • Genes, Reporter
  • Granulosa Cells / metabolism
  • Granulosa Cells / ultrastructure*
  • 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
  • Mammary Glands, Animal / ultrastructure*
  • Mice
  • Microscopy, Electron, Scanning / instrumentation
  • Microscopy, Electron, Scanning / methods*
  • Organoids / metabolism
  • Organoids / ultrastructure
  • Single-Cell Analysis / instrumentation
  • Single-Cell Analysis / methods
  • Staining and Labeling / methods*
  • Theca Cells / metabolism
  • Theca Cells / ultrastructure*
  • Trachea / metabolism
  • Trachea / ultrastructure*
  • Workflow


  • Luminescent Proteins
  • enhanced green fluorescent protein
  • red fluorescent protein
  • Green Fluorescent Proteins