Scanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying

PLoS One. 2021 Jul 7;16(7):e0254165. doi: 10.1371/journal.pone.0254165. eCollection 2021.

Abstract

The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / chemistry*
  • Artifacts
  • Cells, Cultured
  • Desiccation / methods
  • Freeze Drying / methods*
  • Humans
  • Microscopy, Electron, Scanning / methods*
  • Organosilicon Compounds / chemistry*
  • Specimen Handling / methods

Substances

  • Actins
  • Organosilicon Compounds
  • hexamethylsilazane

Grants and funding

MS, ET, MK, FL, DF: Leibniz Institut für neue Materialien (INM), Saarbruecken, Germany: www.leibniz-inm.de MS, FL, DF: Saarland University Saarbrücken, Germany: www.uni-saarland.de MS, ET, FL, DF: Deutsche Forschungsgemeinschaft [CRC 1027 (A10)]. www.dfg.de SP: Ulm University/University Hospital Ulm, Ulm, Germany: www.uniklinik-ulm.de SP: Deutsche Forschungsgemeinschaft [CRC 1149 (A6)]: www.dfg.de The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.