Properties of frozen sections relevant to quantitative microanalysis

Area of Science:

• Materials Science
• Analytical Chemistry
• Microscopy

Background:

• Quantitative X-ray microanalysis of frozen sections faces challenges from ice-crystal and electron-beam damage.
• The reliability of X-ray peak-to-continuum ratios for elemental concentration is debated, particularly concerning ice-crystal scale in dehydrated samples.

Purpose of the Study:

• To re-interpret existing experimental data on X-ray peak-to-continuum ratios in frozen sections.
• To evaluate the impact of ice-crystal scale and electron-beam induced mass loss on quantitative analysis.
• To assess the effectiveness of cold-stage microscopy in mitigating these issues.

Main Methods:

• Re-analysis of experimental data concerning X-ray peak-to-continuum ratios.
• Investigation of electron-beam induced mass loss at varying temperatures (300 K vs. 100 K).
• Assessment of quantitative analysis feasibility at different spatial resolutions (1 micron vs. 100 nm).

Main Results:

• X-ray peak intensities may be affected by ice-crystal scale, but peak-to-continuum ratios are robust after continuum corrections.
• Electron-beam induced mass loss significantly impacts accuracy, but can be substantially reduced using a cold-stage (e.g., at 100 K).
• Quantitative analysis of frozen-hydrated sections at 1-micron resolution is feasible at 100 K, but high-resolution (100 nm) analysis is precluded by mass loss.

Conclusions:

• The peak-to-continuum method remains a viable quantitative technique for elemental analysis in frozen sections, provided continuum corrections are applied.
• Cold-stage microscopy is crucial for minimizing electron-beam induced mass loss, enabling more accurate analysis at moderate resolutions.
• Achieving quantitative X-ray analysis at very high resolutions (sub-100 nm) still presents significant challenges, even with mass measurement techniques.