Cryo automated electron tomography: towards high-resolution reconstructions of plastic-embedded structures

Area of Science:

• Electron microscopy
• Biophysical techniques
• Materials science

Background:

• Automated data collection in electron tomography significantly lowers beam dose.
• Optimizing data collection protocols is crucial for self-consistency and high resolution.
• Understanding beam damage and specimen shrinkage is vital for accurate 3D reconstructions.

Purpose of the Study:

• To develop new protocols for automated electron tomography data collection.
• To quantify beam damage and identify beam-stable embedding media.
• To investigate methods for minimizing specimen shrinkage during data acquisition.

Main Methods:

• Examined effects of irradiation and microwave post-curing on Epon, Epox, and Lowicryl embedding media.
• Quantified beam damage and specimen shrinkage under various conditions.
• Investigated cryogenic data collection to mitigate radiation damage.

Main Results:

• Substantial dose reduction did not significantly decrease specimen shrinkage, even with pre-irradiation.
• Shrinkage is likely caused by stroboscopic illumination patterns in automated collection.
• Embedding resin choice and microwave post-curing had minimal impact on shrinkage.
• Cryogenic data collection with minimal pre-irradiation greatly reduced shrinkage.

Conclusions:

• Automated data collection's illumination patterns can accelerate specimen shrinkage.
• Cryogenic conditions are effective in minimizing radiation damage and shrinkage.
• Low-temperature automated data collection offers a promising approach for high-quality 3D reconstructions.