Cutting-edge technologies and methods for in situ structural biology with cryo-ET

Session

FIB Applications & EM Sample Prep Techniques in Biological Sciences

Authors

Juergen M. Plitzko (1), Oda H. Schioetz (1), Christoph Kaiser (1), Sven Klumpe (1)

Affiliations

1. Max Planck Institute of Biochemistry, CryoEM Technology

Keywords

cryo-focused ion beam milling, cryo-FIB, cryo-lift-out, automation, machine learning, correlative microscopy, cellular structural biology

Abstract text

In recent years, in situ cryo-electron tomography (cryo-ET) has produced many awesome results. In 2016 it gained momentum [1], and since then cryo-focused ion beam milling, or cryo-FIB, has been the method of choice to provide access to cellular material at molecular resolution. The main approach to cell sample preparation by cryo-FIB is lamellae milling, particularly on-the-grid [2]. This encompasses samples that are either simply plunge frozen or more recently for waffles frozen under high pressure [3]. Through the latter method, larger cells or smaller organisms are now amenable since they would exceed the vitrification depth offered by standard plunge freezing. However, waffles thicker than 40 microns are not necessarily suitable for FIB milling with gallium ions. This is where cutting-edge plasma FIB technology can come to one's aid. With higher beam currents and higher ablation rates, even thick material is now accessible within acceptable time scales [4,5]. This is even more true for high-pressure frozen material with a thickness of up to 200 microns, where lift-out is almost obligatory. Yet, cryo-lift-out was introduced as early as 2019 it has remained largely a niche application due to the complexity of the procedure and, more importantly, low success rates [6]. The decisive factors for the success of a method, however, are its user-friendliness, its simplicity and an acceptable or better high throughput. Furthermore, it goes without saying that when one massively reduces the original volume, contextual information becomes crucial, as do options for guiding, localizing, and targeting. This concerns both the on-the-grid lamella milling and, to an even greater extent, lift-out.

We will showcase our latest workflow for in situ structural biology with cryo-ET, covering correlated light microscopy, integrated approaches, automation and machine learning for on-the-grid lamellae milling and cryo-lift-out [7-10]. A new key concept is "serial lift-out," which we designed recently. This method, reminiscent of serial sectioning of embedded material at room temperature, modifies the lift-out process to expose the biology of frozen hydrated material by creating a series of lamellae, yet now at molecular resolution. In addition, we will provide a glimpse into large-scale lamellae and tomogram production, as well as an outlook on revisiting visual proteomics in view of having "1001+" tomograms at hand.

Cryo-FIB technology is clearly a key component of most cryo-ET workflows and has made its way into most structural biology EM laboratories. With further progress, one can imagine that cell biologists will also adopt our somewhat extravagant approach, but only after all the technical hurdles have been overcome.

References

1. Mahamid J, Pfeffer S, Schaffer M, Villa E, Danev R, Kuhn Cuellar L, Förster F, Hyman AA, Plitzko JM, and Baumeister W. 2016. 'Visualizing the molecular sociology at the HeLa cell nuclear periphery', Science, 351: 969-72
2. Rigort A., Bauerlein FJ, Villa E, Eibauer M, Laugks T, Baumeister W, and Plitzko JM. 2012. 'Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography', Proc Natl Acad Sci U S A, 109: 4449-54
3. Kelley K, Raczkowski AM, Klykov O, Pattana Jaroenlak P, Bobe D, Kopylov M, Eng ET, Bhabha G, Potter CS, Carragher B, and Noble AJ. 2022. ‘Waffle Method: A General and Flexible Approach for Improving Throughput in FIB-Milling’ Nature Communications 13(1): 1857
4. Berger C, Dumoux M, Glen T, Yee NBy, Mitchels JM, Patáková Z, Naismith JH, and Grange M. 2022. 'Plasma FIB milling for the determination of structures’ Nature Communications 14:629
5. Dumoux M, Glen T, Ho EML, Perdigão LMA, Klumpe S, Yee NBy, Farmer D, Smith JLR, Lai PYA, Bowles W, Kelley R,  Plitzko JM, Wu L, Basham M, Clare DK, Siebert CA, Darrow MC, Naismith JH, and Grange M. 2023. 'Cryo-plasma FIB/SEM volume imaging of biological specimens', ELife, 10.7554/eLife.83623
6. Schaffer, M., Pfeffer S, Mahamid J, Kleindiek S, Laugks T, Albert S, Engel BD, Rummel A, Smith AJ, Baumeister W, and Plitzko JM. 2019. 'A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue', Nat Methods, 16: 757-62
7. Klumpe S, Fung HKH, Goetz SK, Zagoriy I, Hampoelz B, Zhang X, Erdmann PS, Baumbach J, Müller CW, Beck M, Plitzko JM, and Mahamid J. 2021. 'A modular platform for automated cryo-FIB workflows', Elife, 10: e70506
8. Klumpe S, Kuba J, Schioetz OH, Erdmann PS, Rigort A, and Plitzko JM. 2022. 'Recent Advances in Gas Injection System-Free Cryo-FIB Lift-Out Transfer for Cryo-Electron Tomography of Multicellular Organisms and Tissues', Microscopy Today, 30: 42-47.
9. Smeets M, Bieber A, Capitanio C, Schioetz OH, van der Heijden T, Effting A, Piel W, Lazem B, Erdmann PS, and Plitzko JM. 2021. 'Integrated Cryo-Correlative Microscopy for Targeted Structural Investigation In Situ', Microscopy Today, 29: 20-25.
10. Tacke S, Erdmann PS, Wang Z, Klumpe S, Grange M, Plitzko JM, and Raunser S. 2021. 'A streamlined workflow for automated cryo focused ion beam milling', J Struct Biol, 213: 107743.