The advances in electron cryo-microscopy have enabled high-resolution structural studies of vitrified macromolecular complexes in situ by cryo-electron tomography (cryo-ET). Since utilization of cryo-ET is generally limited to the specimens with thickness < 500 nm, a complex sample preparation protocol to study larger samples such as single eukaryotic cells by cryo-ET was developed and optimized over the last decade. The workflow is based on the preparation of a thin cellular lamella by cryo-focused ion beam milling (cryo-FIBM) from the vitrified cells. The sample preparation protocol is a multi-step process which includes utilization of several high-end instruments and comprises sample manipulation prone to sample deterioration. Here, we present a workflow for preparation of three different model specimens that was optimized to provide high-quality lamellae for cryo-ET or electron diffraction tomography with high reproducibility. Preparation of lamellae from large adherent mammalian cells, small suspension eukaryotic cell line, and protein crystals of intermediate size is described which represents examples of the most frequently studied samples used for cryo-FIBM in life sciences.

CEITEC Masaryk University Brno Czech Republic

See more in PubMed

McMullan G, Faruqi A, Clare D, Henderson R (2014) Comparison of optimal performance at 300keV of three direct electron detectors for use in low dose electron microscopy. Ultramicroscopy 147:156–163 DOI

Zivanov J, Nakane T, Forsberg BO et al (2018) New tools for automated high-resolution cryo-EM structure determination in RELION-3. elife 7:e42166 DOI

Villa E, Schaffer M, Plitzko JM, Baumeister W (2013) Opening windows into the cell: focused-ion-beam milling for cryo-electron tomography. Curr Opin Struct Biol 23:771–777 DOI

Schur FK (2019) Toward high-resolution in situ structural biology with cryo-electron tomography and subtomogram averaging. Curr Opin Struct Biol 58:1–9 DOI

O’Reilly FJ, Xue L, Graziadei A et al (2020) In-cell architecture of an actively transcribing-translating expressome. Science 369:554–557 DOI

Nannenga BL, Gonen T (2014) Protein structure determination by MicroED. Curr Opin Struct Biol 27:24–31 DOI

Nannenga BL, Gonen T (2016) MicroED opens a new era for biological structure determination. Curr Opin Struct Biol 40:128–135 DOI

Al-Amoudi A, Norlen LP, Dubochet J (2004) Cryo-electron microscopy of vitreous sections of native biological cells and tissues. J Struct Biol 148:131–135 DOI

Al-Amoudi A, Studer D, Dubochet J (2005) Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy. J Struct Biol 150:109–121 DOI

Pierson J, Fernández JJ, Bos E et al (2010) Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: electrostatic charging for section attachment and implementation of an anti-contamination glove box. J Struct Biol 169:219–225 DOI

Dubochet J, Zuber B, Eltsov M et al (2007) How to "read" a vitreous section. Methods Cell Biol 79:385–406 DOI

Hsieh C, Schmelzer T, Kishchenko G et al (2014) Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography. J Struct Biol 185:32–41 DOI

Rigort A, Bauerlein FJ, Villa E et al (2012) Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc Natl Acad Sci USA 109:4449–4454 DOI

Schaffer M, Engel BD, Laugks T et al (2015) Cryo-focused ion beam sample preparation for imaging vitreous cells by Cryo-electron tomography. Bio-Protoc 5:e1575 DOI