Electron Optic Documents

The field of Single Particle Analysis (SPA) in cryo-EM has matured to allow for the identification of waters and/or coordinated metal ions in a 3D map of well-behaved proteins, as well as the identification of particulars isoforms of some amino acids¹. Near atomic resolution has been obtained that begins to hint at densities associated with hydrogens². The low signal-to-noise ratio of cryo-EM images as well as the particle mass and the targeted resolution typically requires several hundreds of thousands of individual particle images. The single most time-consuming component in any SPA workflow is positioning the stage on a target hole of a grid. Drift associated with this action becomes increasingly important as the target resolution increases resulting in a natural desire to minimize the frequency of stage positioning. Targeting can alternatively be accomplished using a combination of beam shift with a compensatory image shift to bring the target in the field of view of the camera. This manner of targeting can be achieved with an optional correction for beam tilt. This scheme can be applied to obtain multiple images inside a single hole, or extended across multiple holes (Fig. 1). Here, a 7 x 7 x 8 scheme is depicted yielding 392 images for a single stage movement. Clearly, the ability to increase the reach of the deflectors plays an important role in determining the raw throughput. However, most of the overhead required for imaging goes up on exposure and read-out of the camera.

Fig. 1: Typical multi-hole imaging scheme using 8 images for an intra-hole multi record (left) and a 7 x 7 for an inter-hole multi record yielding a total of 392 images for a single stage move.

JEOL has been steadily working with researchers around the world to improve the throughput of their cryo microscopes, regardless of whether the system has a side-entry goniometer such as a JEM- F200, or whether the scope has an autoloader, i.e. a cartridge- based stage such as the CRYO ARM™ series of microscopes. Using a multi-hole imaging scheme, researchers have been able to reach a hitherto unprecedented milestone of 20,000 images/day on both a CRYO ARM™ 300 II³ and a JEM-F200. Given that many structures on EMPIAR have required around 5000 images, essentially 4-5 projects can be accomplished on a daily basis, which opens up new opportunities for routine high resolution structure determination at unprecedented levels.
 

Fig. 2: Throughput achieved in various years ranging from 2000s to 2021.

References:

1. Merk, A., Fukumura, T., Zhu, X., Darling, J.E., Grisshamer, R., Ognejenović and Subramaniam, S. (2020), IUCrJ 7, 639-643.
2. Tegunov, D., Xue, L., Dienemann, C., Cramer, P. and Mahamid, J., (2021) Nature Methods 18, 186-193.
3. Kinoshita, M., personal communication.