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Summary

Scanning electron microscopy (SEM) sample preparation causes cell distortion. Critical point drying and osmium tetroxide (OsO4) fixation induce boundary retraction, but fibronectin coating can mitigate damage.

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Area of Science:

  • Biological Imaging
  • Microscopy Techniques
  • Cell Biology

Background:

  • Sample preparation is crucial for biological electron microscopy (EM).
  • Quantitative analysis of morphological changes during EM sample preparation is lacking.
  • Scanning electron microscopy (SEM) requires specific sample processing steps.

Purpose of the Study:

  • To quantitatively assess morphological changes during SEM sample preparation.
  • To evaluate the impact of drying methods and osmium tetroxide (OsO4) post-fixation on cell morphology.
  • To identify strategies for reducing cell distortion in SEM.

Main Methods:

  • Correlative superresolution microscopy (SRM) and electron microscopy (EM) were employed.
  • SRM images under hydrated conditions served as a baseline.
  • Morphological changes were measured after chemical drying, critical point drying, and OsO4 post-fixation.

Main Results:

  • Chemical drying and critical point drying caused ~60 nm of cellular boundary retraction.
  • OsO4 post-fixation resulted in an additional ~40 nm of boundary retraction.
  • Coating with fibronectin reduced cell distortion from OsO4 post-fixation.

Conclusions:

  • Drying methods and OsO4 fixation significantly alter cell morphology in SEM preparation.
  • Fibronectin coating is a potential method to minimize OsO4-induced cell distortion.
  • Quantitative data aids in understanding and improving SEM sample preparation protocols.