Freeze-substitution as a preparative technique for immunoelectronmicroscopy: evaluation by atomic force microscopy

Area of Science:

• Biological specimen preparation
• Electron microscopy techniques
• Surface imaging

Background:

• Immunoelectron microscopy requires preserving ultrastructure and antigenicity.
• Osmium tetroxide fixation can obscure antigens, necessitating enhancement methods.
• Atomic force microscopy (AFM) offers high-resolution 3D surface imaging.

Purpose of the Study:

• To evaluate cryofixation and freeze substitution for immunoelectron microscopy.
• To investigate the effects of chemical etching on freeze-substituted tissues for AFM.
• To assess the potential of etching for exposing fine biological structures.

Main Methods:

• Rapid freezing of samples using liquid nitrogen-cooled sapphire block.
• Freeze substitution at -80°C in acetone with osmium tetroxide.
• Embedding in epoxy resin and subsequent chemical etching of sections.
• Analysis of etched sections using atomic force microscopy.

Main Results:

• Cryofixation and freeze substitution preserved excellent ultrastructure.
• Chemical etching improved antigen accessibility but caused structural damage.
• Etching effects varied across tissue components and etching protocols.
• AFM revealed differential removal and collapse of section components.

Conclusions:

• Cryofixation/freeze substitution is suitable for high-quality ultrastructure and antigen localization.
• Chemical etching can expose structures for AFM but requires careful optimization.
• Etching-induced structural alterations need consideration for accurate interpretation.