New principle and robot for vitrification of cryo-EM samples with controllable sample thickness and real-time optical inspection
Koning R.I.¹, Vader H.², van Nugteren M.², Grocutt P.A.², Yang W.³, Renault L.L.R.³, Koster A.J.¹, Kamp A.C.F.², Schwertner M.²
¹Leiden University Medical Center, Electron Microscopy, Cell and Chemical Biology, Leiden, Netherlands
²Linkam Scientific Instruments Ltd., R&D, Salfords, United Kingdom
³NeCen, Leiden University, Institute of Biology, Leiden, Netherlands
Keywords: Vitrification of biological samples, blotting-free ethane plunging, sample preparation robot,cryo-Electron Microscopy (cryo-EM), Single Particle Tomography (SPT), cryo-Electron Tomography (cryo-ET)
INTRODUCTION
Cryo-EM, cryo-tomography, Single Particle Tomography (SPT) and cryo-CLEM (Correlative Light and Electron Microscopy) have become standard tools for the investigation of protein 3D structures, cellular structures and more in the last decade. Despite the progress with cryo-EM instruments and automated sample loading and imaging the routine preparation of cryo-samples can be challenging. Ice layer thickness and uniformity are key parameters for obtaining high quality data.
Cryo-plunging techniques most widely used for vitrification are based on paper-blotting to adjust the thickness of the vitrified layer. We have identified this as a key drawback in the process because the blotting paper soaks up moisture while inside the high-humidity chamber leading to characteristics that are challenging and not always repeatable [2][3].
OBJECTIVES
In this work we aim to address the difficulties of cryo-sample preparation for cryo-EM and cryo-ET by means of automation and by introducing the principle of suction to adjust the film thickness on the grid before plunging.
Materials & methods: comparison with conventional blotting, robot design
Fig 1 compares the traditional cryo sample preparation technique (top row, sample pipetted onto EM grid with support film followed by blotting in humidity chamber) with the method proposed here: sample application via dipping followed by film thickness adjustment via suction (bottom row) [1].
Figure 1: Traditional vs. Cryoplunger workflows
Filter paper used in the traditional technique soaks up moisture from the environment of the humidity chamber, changing properties.
The suction used by our method can be adjusted (flow rate and duration) to control film thickness. It also gives access for direct optical real-time imaging that is used to assess sample conditions and to determine the best moment to trigger plunging [1]. The desktop plunging system is shown in fig. 2, the internal elements in fig. 3.
Figure 2: Cryoplunger
Figure 3: Cryoplunger (internal)
RESULTS
We built a robotic cryo-plunger and demonstrated the use for protein suspensions, lipid vesicles, bacteria and human cells.
See [1] for additional details.
CONCLUSION
We have identified the blotting process in the traditional plunging setups as a key drawback in the process and propose a novel method where fluid is removed for the sample thickness adjustment by means of a calibrated and programmed suction process. Key benefits are repeatability, straightforward automation and direct optical access to the sample for real-time monitoring and control of sample film thickness. The thin-film interference and the liquid surface characteristics observed in the optical microscope give control over the optimisation of film thickness (see [1]). Importantly, the optical image acquired immediately before plunging allows good prediction of sample ice quality – saving valuable EM beam time. The new setup improves repeatability, removes potential risks from manual sample handling and increases speed and throughput via automation.
REFERENCES
[1] Nature Communications, (2022) 13:2985, DOI: 10.1038/s41467-022-30562-7
[2] Nature Methods, VOL 18, May 2021, 463–471, P. J. Peters, et al. DOI: 10.1038/s41592-021-01130-6
[3] J. of Microsc., Vol. 276, Issue 1 2019, pp. 39–45 DOI: 10.1111/jmi.12834