Parallel cryo electron tomography on in situ lamellae

Area of Science:

• Structural biology
• Cryo-electron tomography
• Cellular imaging

Background:

• In situ cryo electron tomography (cryo-ET) is crucial for studying macromolecular complexes in their native cellular environment.
• Current cryo-ET methods face throughput limitations due to sample geometry and serial data acquisition.
• Lamella-shaped samples in cryo-focused ion beam milling restrict the accessible area for tomography.

Purpose of the Study:

• To develop a high-throughput method for in situ cryo electron tomography.
• To increase the speed and data yield from each cryo-focused ion beam milled lamella.
• To enable the collection of tomographic tilt series from previously inaccessible sample areas.

Main Methods:

• Development of the parallel cryo electron tomography (PACE-tomo) method.
• Utilizing a geometrical sample model and optical image shift for parallel data acquisition.
• Recording tens of tilt series simultaneously to improve throughput.

Main Results:

• The PACE-tomo method achieves a throughput faster than 5 minutes per tilt series.
• Demonstrated ability to access sample areas conventionally used for specimen tracking.
• Performance testing confirmed high throughput and data quality with both in vitro and in situ ribosome samples.

Conclusions:

• PACE-tomo significantly enhances the efficiency of in situ cryo electron tomography.
• The method overcomes throughput limitations, maximizing data collection from each sample.
• PACE-tomo is applicable to various microscopes and sample types, advancing structural biology research.