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Learning about truth and biases through experience: section surface corrugation, protein denaturation, and staining
1Institut de génétique et biologie microbienne, IGBM, Université de Lausanne, Switzerland. eduard.kellenberger@igbm.unil.ch
Microscopy Research and Technique
|August 26, 1998
Summary
Thin resin sections for electron microscopy overcome previous biases. Organic liquids do not denature proteins, and resin properties influence antigen accessibility and image resolution, with heavy metal stains being the primary cause of low resolution.
Area of Science:
- Electron Microscopy
- Biochemistry
- Materials Science
Background:
- Conventional electron microscopy preparation methods introduce biases affecting antigenicity and resolution.
- The role of organic solvents and resin properties in antigen accessibility and image quality remains debated.
Purpose of the Study:
- To investigate the validity of current biases in electron microscopy preparation using thin resin sections.
- To evaluate the impact of resin properties and fixation on antigen accessibility and immunolabeling efficiency.
- To identify the primary factors limiting resolution in electron microscopy sections.
Main Methods:
- Development and application of thin resin sectioning techniques for electron microscopy.
- Comparative analysis of protein denaturation by organic liquids.
- Assessment of resin properties (e.g., hydrophobicity, copolymerization strength) and their effect on section surface relief.
- Evaluation of immunolabeling efficiency and image resolution across different preparation conditions.
Main Results:
- No evidence of protein denaturation or loss of antigenicity caused by organic liquids used in resin embedding.
- Resin section surface relief, influenced by copolymerization and fixatives, affects antigen accessibility and immunolabeling.
- Stronger copolymerization leads to flatter relief, improving antigen access but potentially reducing image resolution.
- Heavy metal stains were identified as the main factor limiting section resolution compared to other preparation methods.
Conclusions:
- Thin resin sections offer a method to overcome established biases in electron microscopy sample preparation.
- Resin properties and fixation significantly influence immunolabeling outcomes by modulating surface relief and antigen accessibility.
- Optimizing resin embedding and avoiding heavy metal stains are crucial for enhancing resolution in electron microscopy imaging.