Biological cryo-TEM experiments – Below 3 Å?
The achievable resolution plays an important role in biological cryo-TEM experiments. For a long time it has been impossible to analyze structures smaller 3 Å. The limiting factor was the resolution of the utilized TEM cameras which used to be traditional scintillator-coupled CCD/CMOS cameras. Fig. 1 shows the influence of the development of so-called direct detection cameras on biological cryo-TEM research. It shows the increase over time of the number of publications on the examinations of biological structures in regards to their resolutions at 2, 3 and 4 Å.
In 2011, an experiment that used a direct detection camera was published for the first time. For the experiment, Bridget Carragher and Clint Potter from The Scripps Research Institute used a DE-12 camera from Direct Electron. In the following years, this type of camera gradually replaced traditional CCD cameras, which lead to a rapid increase in the analyses of structures in the range 2-4 Å. Since 2014, it has also been possible to collect structures below 3 Å. An option that had long been considered impossible.
However, since then it has become obvious that resolution is no longer the most crucial factor with biological cryo-TEM experiments.
It is more important to improve the quality of the collected images and to increase the collection speed. Scott Stagg from the Florida State University needed only three days for the acquisition of the structure shown in fig. 2 with a resolution of approx. 2.9 Å. He used camera model DE-20 from Direct Electron mounted on a Titan Krios TEM.
In the past years, Direct Electron has pushed the development of direct detection cameras and currently offers the most advanced camera for biological cryo-TEM experiments: model DE-64. DE-64 is the only direct detection camera with an 8 k x 8 k sensor, which corresponds to a field of view of 67.1 megapixels.
Compared to other direct detection cameras with a 4 k x 4 k sensor, DE-64 has the following advantages:
- Fewer images are needed for single particle analyses.
- Large objects like viruses can be analyzed in their entirety.
- 3D reconstruction is considerably improved by refined CTF fitting.
- A high frame rate enables fast image collection.
The DE-64 features the 10th generation of our revolutionary Direct Detection Device (DDD®) sensor, delivering industry-leading resolution and flexible “movie-mode.” Although building an 8 k direct detection sensor was challenging, Direct Electron did not sacrifice performance by reducing the pixel size or features of the DE-64. The DE-64 has 6.5 μm pixels optimized for superior resolution (MTF) over a broad range of magnifications and exposure rates, it includes an integrated Faraday plate and 2k × 2k survey sensor, and it delivers user-adjustable full-frame streaming at up to 30 frames per second (fps).