Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins

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Abstract

While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300 nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET. In this work, we present a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures. These coating steps eliminate the adverse effects of sample charging on imaging with the Volta phase plate, allowing data acquisition with improved contrast. We discuss optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas that make large areas of unperturbed HeLa and Chlamydomonas cells accessible for cryo-ET at molecular resolution.

Introduction

Single particle analysis by cryo-transmission electron microscopy (TEM) (Frank, 2006) has recently emerged as the method of choice for structure determination of isolated protein complexes (Cheng, 2015, Kuhlbrandt, 2014), enabling the analysis of large, membrane-embedded and flexible macromolecules (Aufderheide et al., 2015, Bartesaghi et al., 2015, Bernecky et al., 2016, Zhou et al., 2015). However, the challenge remains to study the spatial distribution and structural variations of these molecular complexes within their native cellular context (Asano et al., 2015, Mahamid et al., 2016).

Cryo-electron tomography (cryo-ET) provides three-dimensional (3D) views of pleomorphic structures, revealing the menagerie of macromolecules, cytoskeleton and membranes within the cell. In cryo-ET, a tilt-series is acquired by physically tilting and imaging the sample at regular increments, followed by computational reconstruction of these projection images to generate a 3D volume. In order to minimize damage to the specimen, each image in a tilt-series is acquired with low electron dose (∼1 e2). The resulting low signal-to-noise ratio (SNR) is the major constraint for the detection and interpretation of molecular detail within tomograms (Lucic et al., 2013). Recent advances in instrumentation, most notably direct electron detectors, have made it feasible to attain sub-nanometer resolutions with cryo-ET (Pfeffer et al., 2015, Schur et al., 2015). Phase plates, which are add-on devices mounted in the back focal plane of the objective lens, were developed to improve the contrast in images of vitrified cells (Danev and Nagayama, 2010, Glaeser, 2013). The contrast enhancement is generated by an additional phase shift between the scattered and unscattered electron waves at the diffraction plane. A novel type of thin film phase plate, the Volta phase plate (VPP) (Danev et al., 2014), significantly improves the SNR for the low spatial frequencies, enhancing contrast and facilitating high-resolution imaging at minimal or even zero defocus values (Asano et al., 2015, Fukuda et al., 2015, Mahamid et al., 2016).

The attainable resolution in cryo-ET is directly related to sample thickness. In biological samples thicker than 300 nm, electrons are prone to multiple inelastic scattering events, resulting in image blurring and reduced resolution (Grimm et al., 1997). Zero-loss energy filtering can compensate for these adverse effects, but a general thickness limitation remains. Thus, until recently, molecular resolution could only be routinely achieved for small prokaryotic cells (Nans et al., 2015, Ortiz et al., 2006), the thin peripheries of adherent eukaryotic cells (Medalia et al., 2002) and biochemically isolated organelles (Daum et al., 2010, Davies et al., 2011, Li et al., 2012, Pfeffer et al., 2015, Pigino et al., 2011, von Appen et al., 2015). With the emergence of cryo-FIB thinning of frozen-hydrated cells (Marko et al., 2007, Rigort et al., 2010b), a wide variety of cell types ranging from larger bacteria to neuronal primary cultures have been made accessible for cryo-ET (Engel et al., 2015b; Fukuda et al., 2015, Hagen et al., 2015, Mahamid et al., 2016, Rigort et al., 2012, Strunk et al., 2012). Ongoing developments hold promise for targeting specific cellular structures (Arnold et al., 2016), even within tissues and multicellular organisms (Hsieh et al., 2014, Mahamid et al., 2015). While FIB micromachining methods allow access to the vast majority of eukaryotic cellular structures, the resulting lamellas of “free-standing” biological material have little contact to the conductive TEM grid. This increases the likelihood of beam-induced specimen movement during TEM imaging. Additionally, electrostatic charging of the specimen can alter the electron wavefront, compromising the use of phase plates (Danev and Nagayama, 2010, Mahamid et al., 2016). Fine metal coating of the final FIB lamella was demonstrated to render the lamellas conductive, allowing the acquisition of tomographic data in combination with the VPP (Mahamid et al., 2016).

Here, we describe a cryo-sample preparation workflow for in situ cellular tomography. We demonstrate how to reproducibly prepare lamellas that exhibit the features required to access molecular-resolution information over large areas (up to 700 μm2). These lamellas must be properly vitrified, homogenous in thickness and free of mechanical deformations and surface contamination. To produce such lamellas, the optimized preparation procedure counteracts specimen charging by employing conductive metal sputtering and prevents typical FIB preparation artefacts such as preferential milling and curtaining by additional deposition and milling strategies. Charging during VPP imaging is prevented by coating the final lamellas with a fine conductive metal layer. We demonstrate the application of this workflow to thinning HeLa cells and the photosynthetic single-celled alga, Chlamydomonas reinhardtii. The reconstructed 3D tomographic volumes reveal that macromolecular complexes, including those embedded in lipid membranes, can be directly visualized by in situ cryo-ET.

Section snippets

Sample preparation workflow

In this section, we describe a routine for FIB milling of frozen-hydrated cells to obtain thin, conductive lamellas that are suitable for VPP-assisted cryo-ET (Fig. 1). We have previously applied this workflow to HeLa cell cultures (Mahamid et al., 2016). We begin with a brief introduction of the preparation procedures, followed by discussion of the underlying methodological details.

A basic requirement for cryo-FIB lamella preparation is a vitrified biological specimen. Incomplete vitrification

Discussion

The fidelity with which macromolecules can be visually identified in cryo-electron tomograms is dependent on several factors. Foremost is the thickness of the sample volume. Resolution scales with thickness for any TEM investigation. However, a compromise has to be found between what details one would like to see and how big the final tomographic volume should be.

The ability to detect and identify molecular complexes is not limited by thickness alone. The characteristic material properties of

Cell culture

HeLa cells stably expressing beta-tubulin:GFP off a BAC and H2B:mCherry off a plasmid were cultured in the presence of G-418 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine and maintained using standard procedures. Cell cycle synchronization was achieved by an initial block using 2 mM thymidine (Sigma-Aldrich Chemie, Munich, Germany) for 18 h, release for 6 h, followed by a second block using 2 μm S-Trityl-l-Cysteine (Enzo Life Sciences, Loerrach,

Acknowledgements

We are grateful to Radostin Danev for assistance with VPP operation, to the department’s workshop for the design and production of various tools, and to Dimitry Tegunov for the development of the frame-alignment tool. M.S. was supported by the European Commission grant agreement ERC-2012-SyG_318987-ToPAG. J.M. was supported by postdoctoral research fellowships from EMBO and HFSP, and by the Weizmann Institute Women in Science Program. B.E. was supported by a postdoctoral research fellowship

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