Application of high-resolution scanning electron microscopy to biological macromolecules

Area of Science:

• Biophysics
• Microscopy
• Structural Biology

Background:

• Ultrahigh-resolution scanning electron microscopes (SEMs) offer potential for observing biological macromolecules.
• Existing preparation methods are inadequate for many molecules, especially those with low contrast or high sensitivity to electron beam damage.
• Improved sample preparation is crucial for realizing the full potential of advanced SEM technology.

Purpose of the Study:

• To establish effective preparation methods for imaging biological macromolecules using ultrahigh-resolution SEM.
• To overcome limitations of current techniques, such as poor contrast and electron beam sensitivity.
• To demonstrate the feasibility of visualizing diverse macromolecules with advanced SEM.

Main Methods:

• Application of heavy-metal impregnation techniques: phosphotungstic acid, uranyl acetate, and osmium tetroxide mordanted by tannic acid.
• Optimization of SEM conditions: improved vacuum in the specimen chamber and use of a heated specimen stage to reduce contamination.
• Imaging of specific biological macromolecules including haemocyanin, ferritin, apoferritin, thyroglobulin, and immunoglobulin M.

Main Results:

• Successful imaging of haemocyanin, ferritin, apoferritin, thyroglobulin, and immunoglobulin M using the developed preparation methods.
• Demonstration that heavy-metal impregnation enhances contrast and protects against electron beam damage.
• Validation of improved vacuum and heated stage for minimizing specimen contamination during imaging.

Conclusions:

• Developed heavy-metal impregnation methods combined with optimized SEM conditions enable high-resolution imaging of biological macromolecules.
• Ultrahigh-resolution SEM is a powerful and promising tool for studying the morphology of biological macromolecules.
• This approach expands the scope of macromolecular structural analysis using electron microscopy.