Freeze-substitution as a preparation technique for biological X-ray microanalysis

Area of Science:

• Materials Science
• Biological Sciences
• Analytical Chemistry

Background:

• Preserving diffusible elements in biological samples is crucial for accurate analysis.
• Conventional methods often lead to element translocation or loss.
• Developing reliable techniques for elemental analysis in situ is essential.

Purpose of the Study:

• To present a freeze substitution method for preserving diffusible elements.
• To enable quantitative elemental analysis in biological tissues using STEM.
• To establish a robust method for mass-per-unit-volume determination.

Main Methods:

• Freeze substitution using an acrolein-ether solvent under anhydrous conditions.
• Embedding specimens in epoxy or methacrylate resins.
• Sectioning embedded samples and imaging using scanning transmission electron microscopy (STEM).

Main Results:

• Virtually total retention of diffusible elements was achieved.
• In situ elemental retention was observed in organ lumina and body fluid spaces.
• Accurate mass-per-unit-volume analysis was enabled by precise section thickness determination.
• Monitoring of charging, drift, mass loss, contamination, and beam current was facilitated.

Conclusions:

• The described freeze substitution and embedding technique effectively preserves diffusible elements.
• This method supports quantitative elemental analysis in biological specimens via STEM.
• The technique offers a reliable approach for mass-per-unit-volume determination, improving analytical accuracy.