Fluorescence microscopy of single viral capsids.

Area of Science:

• Biophysics
• Molecular Biology
• Microscopy

Background:

• Single-molecule fluorescence microscopy is crucial for understanding protein complexes.
• Bacteriophage T7 capsids offer a model system for studying molecular interactions.
• Controlling molecular motion is essential for high-resolution imaging.

Purpose of the Study:

• To establish a foundation for single-molecule fluorescence microscopy of protein complexes.
• To achieve fluorescence microscopy of individual, nucleic acid-free bacteriophage T7 capsids.
• To demonstrate the manipulation and analysis of single protein and DNA molecules.

Main Methods:

• Staining bacteriophage T7 capsids with Alexa 488 (green emission).
• Embedding capsids in agarose gel to control three-dimensional thermal motion.
• Visualizing single, diffusing T7 capsids alongside gel-embedded, ethidium-stained DNA molecules.

Main Results:

• Agarose gel embedding significantly reduced and controlled capsid thermal motion.
• A linear relationship was observed between gel concentration and effective diffusion constant.
• Successful simultaneous visualization of single protein capsids and DNA molecules was achieved.

Conclusions:

• Single-molecule analysis of protein and DNA interactions is feasible.
• This technique provides a basis for studying DNA packaging and metabolism.
• Maintaining biochemical activity remains a key challenge for future research.