Next generation sample preparation for cryo-ET – instruments, accessories and workflows

Abstract text (incl. figure legends and references)

The recent technological breakthrough in cryo-electron tomography (cryo-ET) opens the possibility of imaging macromolecular complexes in their native cellular context at sub-5Å resolution [1,2]. This technology is the foundation for an emerging field in the life sciences, often termed structural cell biology, capable of revealing how different conformational states of protein complexes are linked to biological functions. However, imaging is highly dependent on sample preparation. This is especially true for transmission electron microscopy (TEM), where samples must be thin enough to provide high-resolution information, homogeneous in thickness for consistent high-quality data, and in their original state, as in cryo-TEM, to preserve the structural features of macromolecules. Sample preparation, while largely underappreciated, is thus a vital part of imaging.

Since its beginnings as a sample-thinning technique for cryoET, cryogenic focused ion beam milling (cryoFIB) using gallium ions has evolved into a procedure that encompasses several different methods. Methods like on-the-grid lamella-preparation of plunge frozen samples, iterative FIB ablation and scanning electron microscopy (SEM) imaging, and most recently lift-out for high-pressure frozen (HPF) multicellular samples [3,4]. The latter is an established method in materials science, but since its introduction for cryo-ET, it has been used rather rarely [5]. The reasons why the lift-out method falls short compared to lamellar preparation on the grid are quite obvious: First, it is very time consuming to mill high-pressure frozen (HPF) samples that are up to 200 microns thick; second, the whole process is often disappointingly inefficient, mainly because of its complexity; third, there is no way to assess ice quality before or during preparation, only after the first TEM image does one know whether the ice is "good" or "bad"; and finally, the "survival rate" of lift-out lamellae is low, either they are lost during manual transfer or they are contaminated beyond recognition.

Yet, there must be ways to streamline this process and make multicellular specimens and small organisms amenable to routine access in a reasonable time frame. PlasmaFIB technology promises to do just that: Higher beam currents and higher ablation rates to make bulky samples easily accessible, hardware automation that frees the user from any manual operation and increases throughput and survival rates, and finally integrated solutions for targeting (correlative fluorescence microscopy, CLEM) and ice quality assessment.

Here we present the latest developments towards full automation for plunge-frozen and high pressure frozen samples, as well as initial results and experiences with a novel plasma cryo-FIB system specifically designed for large scale cryo-FIB milling.

References:

[1] D. Tegunov et al., Nature Methods 18, 186–193 (2021).

[2] S. Khavnekar et al. bioRxiv; DOI: 10.1101/2022.06.16.496417 (2022).

[3] S. Klumpe et al., eLife 2021;10:e70506 (2021).

[4] S. Klumpe et al., Microscopy Today 30(1), 42-47 (2022).

[5] M. Schaffer et al., Nature Methods 16, 757-762 (2019).