Electron tomography of ice-embedded prokaryotic cells

Area of Science:

• Microbiology
• Structural Biology
• Electron Microscopy

Background:

• Archaea are single-celled microorganisms with unique cellular structures.
• Understanding archaeal cell architecture is crucial for various biological processes.
• Vitreous ice embedding and electron tomography are advanced techniques for cellular imaging.

Purpose of the Study:

• To investigate the 3D structure of whole archaeal cells using automated energy-filtered electron tomography.
• To determine the achievable resolution for imaging archaea cells of varying thicknesses.
• To compare experimental results with theoretical predictions for electron tomography.

Main Methods:

• Whole archaea cells were embedded in vitreous ice using plunge freezing.
• Automated energy-filtered electron tomography was performed at 120 kV.
• Tilt series of images were acquired and reconstructed to visualize cellular structures.

Main Results:

• Reconstructed structures achieved a resolution of 20-40 nm for cells 300-750 nm thick.
• Individual S-layer stalks of Pyrobaculum aerophilum were visualized in 3D.
• Theoretical analysis indicated that specimen thickness significantly impacts attainable resolution.

Conclusions:

• Low-dose electron tomography can resolve fine structures like S-layers in archaea.
• Higher accelerating voltages (300 kV) are beneficial for thicker specimens (>100 nm).
• Experimental resolutions currently lag behind theoretical limits by a factor of 2-5.