Enabling a routine approach to CryoEM sample optimization

Paul Thaw¹ , Michele C. Darrow¹, Tim Booth¹, John P. Moore¹, Klaus Doering¹, Russell S. King¹

¹ SPT Labtech LTD., Melbourn Science Park, Hertfordshire, UK

In the cryogenic electron microscopy (cryoEM) workflow, improvements in microscope stability, direct detectors and image processing have enabled high throughput data collection strategies and shifted the remaining bottleneck to sample quality. The process of obtaining a film of vitreous ice of an appropriate thickness, with evenly distributed particles is for many not straightforward. To address the limits of achievable resolution due to the ubiquitous effects of the air water interface (AWI) [1] conventional sample preparation methods use iterative cycles of trial-and-error optimization experiments and screening routines to search untraceable data for favourable outcomes [2]. When it fails, the abundance of low-quality samples causes downstream inefficiencies such as the brute force data collection methods used to overcome poor sample quality, serving only to pressurize the microscope and computing resources. This results in a failure to achieve early research milestones critical for funding and resource allocations and while excessively large data sets may improve outcomes for a few samples, they do not tackle the root-cause of the issue, namely poorly behaving samples.

We have developed the chameleon system with the goal of moving towards the more reproducible outcomes and walk-up usability required to democratize the sample preparation workflow. Ideally, researchers require the ability to freeze where and when the biochemistry is carried out when pursuing cutting-edge targets, placing the requirement for change squarely on modernizing the sample preparation workflow to provide quality results, and reduce training burdens.

The chameleon system is a blot-free, pico-litre dispense instrument that enables high-speed plunging (as fast as 54ms) to robustly freeze and optimize samples for use in cryoEM [1]. The chameleon system was developed from Spotiton [2,3] and uses self-wicking copper nanowire grids to control the formation of the thin sample film [4] ‘on-the-fly’ as the grid passes in front of the dispenser on its way to the cryogen bowl, Automated procedures and intuitive workflows allow users to optimize plunge times and ice thicknesses to understand and then mitigate negative sample specific AWI effects for each unique project. Workflow examples and methods will be discussed alongside sample outcomes from chameleon users in the field.

[1] A Noble, et al. Nature Methods 15 (2018), p. 793-795

[2] B. Carragher, et al. J. Microsc 276 (2019), p. 39-45.

[3] MC Darrow, et al. Microscopy and Microanalysis, 25 (2019), p. 994-995

[4] I Razinkov, et al. Journal of Structural Biology 195 (2016), p. 190-198