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Avoiding gas bubble formation during freeze substitution.

D B Fisher1

  • 1Department of Botany, Washington State University, Pullman 99164-4238.

Biotechnic & Histochemistry : Official Publication of the Biological Stain Commission
|January 1, 1991
PubMed
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Gas bubbles form during freeze substitution due to carbon dioxide solubility. Transferring tissues to chilled solvent before warming minimizes bubble formation, preserving sample integrity.

Area of Science:

  • Cryo-EM and Electron Microscopy Sample Preparation

Background:

  • Freeze substitution is a critical technique for preserving biological tissues.
  • Gas bubble formation during warming is a common artifact in freeze substitution.
  • Carbon dioxide (CO2) solubility in substitution solvents contributes to bubble formation.

Purpose of the Study:

  • To identify the cause of gas bubble formation during freeze substitution.
  • To propose a method for minimizing gas bubbles in tissue samples.

Main Methods:

  • Investigating the role of carbon dioxide solubility in freeze substitution artifacts.
  • Implementing a modified warming protocol involving chilled solvent transfer.

Main Results:

  • Gas bubbles frequently form when tissues are warmed to room temperature after freeze substitution.

Related Experiment Videos

  • The extreme solubility of CO2 in the substitution solvent is identified as the primary cause.
  • Briefly transferring tissues to freshly chilled solvent before warming significantly reduces bubble formation.

    • Conclusions:

    • Gas bubble formation during freeze substitution is primarily caused by CO2 solubility.
    • A simple protocol modification can effectively minimize this artifact.
    • This method aids in preserving sample integrity for high-resolution imaging.