Rapid virus subunit visualization by direct sedimentation of samples on electron microscope grids.

Area of Science:

• Virology
• Microscopy
• Biochemistry

Background:

• Ultracentrifugation is crucial for isolating and concentrating viral particles.
• Electron microscopy (EM) requires concentrated samples for detailed structural analysis.
• Current methods for viral sample preparation for EM can be time-consuming.

Purpose of the Study:

• To evaluate direct ultracentrifugation using the Airfuge for rapid concentration of viral samples on EM grids.
• To determine the sensitivity enhancement offered by this technique compared to traditional methods.
• To assess the preservation of viral structure and subunits after ultracentrifugation.

Main Methods:

• Direct ultracentrifugation of viral samples (up to 240 microliters) on EM grids using an A-100 fixed angle rotor (30 degrees, K=19, 95,000 rpm).
• Sensitivity assessment using latex spheres to compare with the inverted drop procedure.
• Application to rubella virus and respiratory syncytial virus (RSV) associated subunits.

Main Results:

• Achieved 10- to 40-fold increase in sensitivity for viral particle detection, dependent on particle size.
• Reduced observation time for EM analysis.
• Improved visualization of viral membrane subunits and immuno-stimulating complexes (ISCOMs).
• Demonstrated preservation of morphology for rubella virus and RSV subunits.

Conclusions:

• Airfuge direct ultracentrifugation is a rapid and effective method for concentrating viral samples for EM.
• This technique significantly enhances sensitivity and improves the visualization of viral structures and their components.
• The method preserves the integrity of viral particles and subunits, facilitating detailed structural studies.