Optimising the tilt increment for in situ cryo-electron tomography

Cryo-electron tomography, or cryo-ET, allows for high-resolution, three-dimensional imaging of cellular structures while they are frozen in their natural state. However, the quality of the images is limited by a trade-off between the angle at which the images are taken and the damage caused by the radiation used to capture them.

The angle at which the images are taken, known as the angular increment, is a crucial factor that influences the quality of the final tomogram and the subsequent analyses. Determining the optimal angle increment is difficult due to the high demands on the microscope time, storage requirements, and computational resources.

In this study, the researchers investigated the effects of different tilt increments on the quality of cryo-ET images. They examined tilt increments of 1°, 2°, 3°, 5°, and 10° using lamellae, or thin slices, from Dictyostelium discoideum cells, a type of slime mold. By using lamellae from the same type of cells, the researchers aimed to minimize variations in the cellular structures and focus solely on the impact of the tilt increment.

The study found that using finer tilt increments, such as 1° or 3°, resulted in more well-aligned tomograms. These tomograms had higher signal-to-noise ratios, which means they were clearer and more easily interpretable. Furthermore, the finer tilt increments led to better performance in template matching and subtomogram averaging.

Template matching is a process used to identify and classify similar structures across multiple images, while subtomogram averaging involves combining multiple low-resolution images to create a higher-resolution image of a specific structure.

After analyzing the data collected from the different tilt increments, the researchers concluded that a tilt increment of 3° provided the best balance between image quality, alignment accuracy, the amount of radiation damage per image, and the efficiency of the processing required to create the final tomogram. This finding offers practical guidance for both high-throughput and high-resolution cryo-ET studies.

By choosing a 3° tilt increment, researchers can optimize their data collection strategies to achieve the best possible results while minimizing the risks associated with radiation damage. This recommendation can help shape future approaches to cryo-ET data acquisition, ultimately advancing our understanding of cellular structures at an unprecedented level of detail.