Published: November 20th, 2021
We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the Saccharomyces cerevisiae.
Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.
Recent technological and software developments have made electron cryo-microscopy (cryo-EM) of vitrified biological specimens one of the key techniques in structural biology research in the last decade1,2. The preparation of a specimen for cryo-EM usually consists of the application of a purified protein or a complex of protein with nucleic acid onto the sample carrier (TEM grid), followed by removal of most of the liquid with a filter paper, and plunge freezing of the grid with the residual thin layer of a sample into liquid ethane or propane3. The sample is thus fixed in a thin layer (typically <80 nm) of amorphous buffer in a fully hydrated state, at near-native conditions, and without need of any chemical fixation or heavy metal contrasting. Imaging of the structurally homogeneous specimen in the transmission electron microscope then results in data that can be used for the determination of the three-dimensional structure of the macromolecule at near-atomic resolution using a single particle analysis protocol2. Such an in vitro structure corresponds to the representation of the macromolecule under the conditions and treatment utilized during sample preparation. Although the structures determined under the in vitro conditions usually correspond to the fully functional state of the macromolecule, the capacity to image spatial relationships among various macromolecules inside the cell would provide an additional functional context to the structural data.
Cryo-electron tomography (cryo-ET) is used to reconstruct 3D volumes of pleomorphic objects or macromolecular complexes in situ4,5. The advantage of cryo-ET is that the three-dimensional information is obtained by imaging a single entity. However, the resolution at which individual macromolecular complexes or organelles are observed is very limited. Therefore, averaging of the macromolecules (sub-tomogram averaging, STA) with the same structure from a larger number of tomograms is necessary to reach 4-8 Å resolution models from the cryo-ET data6,7. It has recently been shown that cryo-ET and STA can also be applied to determine high-resolution structures of macromolecular machines such as ribosomes in the context of the cellular environment7. However, the utilization of transmission electron microscopy is limited by the specimen thickness. In general, this is not a problem for single-particle cryo-EM where optimization of the vitrification conditions can eventually result in the embedding of the sample in a thin layer of ice. On the other hand, most of the cells are not in fact electron transparent for the 300 keV electron beam. The inelastic mean free path in the vitrified biological specimens for the 300 keV electrons is approximately 395 nm8, which means that cryo-ET studies are limited to the cellular periphery for most of the cells.
Different techniques were developed to thin the sample down to a sufficient thickness for cryo-ET. Cryo-ultramicrotomy utilizes mechanical slicing of the sample with a diamond knife at the liquid nitrogen temperature (-196 °C) to provide 60-80 nm thick sections suitable for cryo-ET9,10,11. Multiple sections can be prepared from a single cell and the data analysis can eventually produce 3D structural information for the larger part of the cell. However, the mechanical slicing can cause several artifacts such as curved sections, crevasses, or sample compression, which may influence the resulting structure and bias the cryo-ET data10,11,12. Cryo-focused ion beam micromachining (cryo-FIBM) represents an alternative approach where a thin cellular section is prepared by gradual ablation of the specimen using a focused beam of Ga+ ions (FIB) in a multi-step process, which can result in 80–300 nm thick cellular cross-section (lamella)13,14,15. In contrast to cryo-ultramicrotomy, only one lamella is prepared from a single cell, which represents ~0.3–3% of its volume, and micromachining of multiple cells is usually necessary to find a region of interest in the milled cross-section. In addition, the throughput of the whole workflow is nowadays still fairly low, often limited to 6–8 high-quality lamellae from an 8-hour cryo-FIBM session. On the other hand, the cryo-FIBM cross-sections are devoid of any compression artifacts and provide suitable input for high-resolution cryo-ET. Moreover, the transfer of the lamella to the sample carrier for cryo-ET is not necessary as the sample is retained on the TEM grid during the whole lamella preparation process and the same grid can be subsequently transferred to TEM. We expect that the throughput of the cryo-FIBM will be significantly improved soon, primarily from availability of software for unsupervised lamella milling16,17 and utilization of FIBs operating on the principle of charge-couple plasma, which will afford faster material ablation.
Saccharomyces cerevisiae (yeast) are eukaryotic cells of spherical shape and diameter of ~2-5 µm. Thanks to its size, accessibility, genetics, generation time, and simple manipulation, the yeast is extensively studied as a eukaryotic model organism in cellular and molecular biology research similar to Escherichia coli, which is well studied as a prokaryotic model organism in bacteriology. The yeast can be easily cultured in suspension and a high quantity of cells is generated in a short time (doubling time 1 – 2 hours). More importantly, yeast shares a complex internal cellular structure with animal and plant cells while retaining a small genome comprised of a low content of non-coding DNA. Structural characterization of the yeast proteome from the high-resolution in situ data can thus help to provide a mechanistic description for the extensive amount of functional data available in the literature.
Herein, we provide a comprehensive protocol for the acquisition of in situ cryo-ET data on the yeast sample, which covers all steps from the sample cultivation down to the cryo-FIBM lamella preparation, and the specimen transfer to TEM for cryo-ET data acquisition.
1. Cultivation and preparation of Saccharomyces cerevisiae cells for vitrification
2. Vitrification of the Saccharomyces cerevisiae specimen
3. Mounting TEM grids into the grid cartridge
NOTE: The workflow described here utilizes the TEM grids mounted into the grid cartridge to facilitate sample handling and transfer between SEM and TEM microscopes. The cartridge assembly consists of a C-ring, a TEM grid, and a C-clip ring. Other options are available when working with instrumentation from other microscope manufacturers. The grid cartridge assembly workstation is filled with LN2. The LN2 level covers the grid box with the TEM grids, but the mounting of the TEM grids into the cartridge is performed in LN2 vapors. It is highly recommended to wear a protective facemask or shield during the vitrification procedure to prevent contamination from breathing to the specimen. Do not work with the tools that have accumulated ice contamination.
4. Loading and manipulation of the sample to the FIB-SEM microscope
NOTE: The instructions were written for the utilization of the dual-beam microscope Versa 3D equipped with the PP3010 cryo-FIB/SEM preparation system. Alternative solutions may require different specific parameters; however, the general concept of the workflow should be still valid.
5. Preparation of the Saccharomyces cerevisiae lamella
6. Transfer of Saccharomyces cerevisiae lamella to cryo-TEM
The Saccharomyces cerevisiae culture was harvested in the middle of the exponential growth phase. We prepared two types of specimens in which the cells were either distributed as small clusters of several cells over the surface of the TEM grid (Figure 1A,C) or formed a continuous monolayer over individual grid squares of the TEM grid (Figure 1B,D). The discriminative factor for the preparation of the sample with either distinct cell islets or the cellular monolayer is the concentration of the cell culture applied to the TEM grid. The harvested cell culture was concentrated to OD600 = 1.0 for the former case, or to OD600 = 30 to 60 for the latter case, respectively. The sample for the preparation of the cellular monolayer was further supplemented with 5% v/v glycerol prior to vitrification. The glycerol is critical for the vitrification of the buffer solution, which fills the space in between the cells (Figure 2) as the reflections from the crystalline buffer can be detrimental for proper positional tracking and focusing during cryo-ET data collection.
In addition, the yeast suspension culture was blotted against the non-absorbent material such as the PTFE blotting pad or the custom 3D printed pad made of FlexFill 98A material. The blotting paper was positioned only on the backside of the grid with respect to the sample application (back-blotting). The back-blotting strategy is recommended for the suspension culture plunge freezing as blotting with the filter paper from both sides results in adhesion of the cells to the blotting paper (Figure 1E).
The protocol described here utilizes TEM grids clipped in the grid cartridge, which forms stable support for the grid and facilitates sample handling of the sample after the vitrification. This enforces a necessity that other sample holders and shuttles in FIB/SEM and TEM microscope can accept such a grid cartridge.
Upon transfer of the sample into the the FIB/SEM microscope, the specimen was first coated with a 0.3–1.0 μm layer of methylcyclopentadienyl platinum using the microscope gas injection system (GIS). An additional layer of the inorganic iridium was sputtered on the sample surface to harden the GIS layer and render the surface conductive. The lamellae were milled in multiple steps (Figure 3) where (I) the milling current, (II) the lamella width, and (III) the distance of the milling area above and below the samples were decreased in a stepwise manner. The final milling step (“polishing”) was carried out at low current (10-30 pA) only from the top side of the lamella and with the sample inclined by an additional 1° towards the Ga+ beam. Utilization of the described protocol has on average resulted in 8-10 lamellae prepared on two TEM grids within one 6-8 hour session.
The TEM grids with the lamellae were subsequently transferred into a transmission electron microscope. The lamellae were first screened and only those which showed minimal curtaining (artefacts stemming from uneven milling across the lamella surface), low surface contamination level, and good cellular contrast (usually observed for lamellae with <200 nm thickness) were selected for the acquisition of the cryo-ET data. In addition, lamellae containing cracks across the whole length were discarded from data collection. In general, about 50% of the lamellae transferred to TEM were suitable for data acquisition. Tilt series were collected on the post-GIF K2 direct electron detector with the energy-selecting slit set to 20 eV. The data collection was carried out in SerialEM software18 and the tilt series were collected using a dose symmetric scheme19 with the tilt range of ±60° and the increment of 3°. The data was acquired at the magnification corresponding to the pixel size of 3.47 A/px. The overall dose of 65 e/Å2 was uniformly distributed over the individual sub-frames. The tilt images were collected as a set of three frames, which were subsequently corrected for the motion and radiation damage during data acquisition using MotionCor220 program. Parameters of the contrast transfer function were estimated using Ctffind421. The tilt series were processed in eTomo18. The patch tracking routine was used to align the images. The tomogram was reconstructed using a weighted back projection algorithm after 2x binning of the images, and subsequently filtered using SIRT-like filter (set to 8 iterations) in IMOD18. The tomogram segmentation was carried out manually in Amira software22. The reconstructed tomograms provide a high-resolution representation of the yeast cellular interior and enable us to observe organelles such as vacuoles or mitochondria at a high level of detail or study macromolecular complexes such as microtubules, or nuclear pore complexes in situ and under near-native conditions (Figure 4).
Figure 1: FIB and SEM images of vitrified S. cerevisiae
FIB (A) and SEM (C) images of the small yeast clusters vitrified on the TEM grid. FIB (B) and SEM (D) images of the yeast forming a continuous monolayer on the grid surface. The sample was coated with GIS and Irridium layer before imaging. The scale bars in panels A-B corresponds to 10 μm. The yeast sample is blotted against non-absorbent material such as PTFE or FlexFill 98A (green) and with the blotting paper positioned from the backside of the grid (white, E). Please click here to view a larger version of this figure.
Figure 2: S. cerevisiae lamellae
A TEM image of a lamella micromachined from the sample with continuous monolayer yeast over the grid surface. The reflections observed between the cells contained improperly vitrified medium/buffer (A, highlighted with red circles). A TEM image of lamella generated on the yeast vitrified into a continuous monolayer with the addition of 5% glycerol into the medium/buffer (B). Scale bar corresponds to 2 μm. Please click here to view a larger version of this figure.
Figure 3: Cryo-FIBM workflow
Schematic depiction of the lamella milling process. The initial rough milling steps are performed at high FIB currents from both sides of the tentative lamella position (highlighted in green) whereas the final polishing step is performed only from the top side and at low FIB current (highlighted in orange, see accompanying video at 6:33). Please click here to view a larger version of this figure.
Figure 4: Yeast organelles and macromolecular complexes depicted by cryo-ET
Slices of the reconstructed tomograms depicting a vacuole (A, scale bar: 200 nm), ribosomes (B, scale bar: 200 nm), a paracrystalline core of peroxisome (C, scale bar: 100 nm), microtubule (white arrow) in the proximity of unidentified fibrous structure (black arrow, D, scale bar: 100 nm), details of multiple microtubules (E, scale bar: 50 nm), a nuclear membrane with pores indicated by arrows (F, scale bar 200nm), mitochondrion (G,H, scale bar: 100 nm, the arrows indicate individual cristae), a bundle of unidentified filamentous structures (I, scale bar: 100 nm). Panels B, C, D, E, G contain a section of tomograms prepared from small clusters of cells whereas the sections of tomograms collected on lamellae from a monolayer of the cells are shown in panels A, F, H, I. Please click here to view a larger version of this figure.
The preparation of the cellular samples for cryo-ET is a complex workflow that requires utilization of several high-end instruments. The sample quality can be compromised during each preparation step that influences the throughput of the whole protocol. In addition, the necessity of the sample transfer between individual instruments poses an additional risk of sample contamination or devitrification. Therefore, optimization of individual steps in the sample preparation workflow is of high importance to increase the throughput and reproducibility of the lamella preparation workflow. The protocol presented here describes the optimized preparation of Saccharomyces cerevisiae for structural characterization of macromolecular complexes in situ by cryo-ET.
The protocol describes the preparation of two types of yeast specimens that mainly differ in the concentration of the cells on the TEM grid. Both yeast samples yielded high-quality lamellae for cryo-ET and selection of the specimen type can be made in accordance with the goals of the particular study. The yeast forms isolated clusters of few cells randomly scattered over the grid surface in the first case, whereas a continuous monolayer of cells is present on the TEM grid surface for the second sample type. The former is suitable for fast lamella preparation thanks to the small volume of the material that must be milled away. The final lamella is fairly short, and, therefore, contains only 2-4 cellular cross-sections. The areas suitable for sample preparation are randomly distributed over the grid surface including the grid squares, which may partially restrict the automation of the lamella preparation workflow. The latter type of the specimen requires utilization of larger currents during the initial milling phase to retain the overall milling time. In addition, this type of sample is more prone to artifacts that stem from uneven milling (curtaining). Therefore, GIS is sputtered onto the sample surface for a 50% longer period than in the case of the sample with small cellular clusters to form a thicker protective layer. Next, the sample is sputtered with an additional layer of Iridium (alternatively platinum or gold) to cure the GIS layer, render it stiffer, and increase the sample surface conductivity. FIBM of additional areas on each side of the lamella (~2-5 μm from the lamella edge) during the first step of the rough lamella milling was found beneficial to diminish the number of broken lamellae most probably due to decreased tension in the final cross-section23. The final lamella is long and contains ~10 cellular cross-sections, which increases the number of regions suitable for cryo-ET. The improper vitrification of the medium or buffer between the cells can be easily attenuated by the addition of the cryo-protectant to the buffer solution (5% glycerol used in this study). Since most of the squares are suitable for lamella preparation, the sample with cells organized into a continuous monolayer is well suited for unsupervised lamella preparation.
Another important aspect in the lamella preparation workflow is the transfer to the transmission electron microscope and proper positioning of the lamella to the microscope stage tilt axis. Optimally, the lamella main axis is perpendicular to the tilt axis of the microscope which affords tracking and focusing at the height of the imaged region and prevents the lamella edges from shielding the field of view at high tilt angles. When collecting the cryo-ET data using the dose-symmetric scheme,18 the sample should be initially rotated in the microscope to compensate for the tilt of the lamella with respect to the grid plane.
The authors have nothing to disclose.
This work was funded by the Technology Agency of the Czech Republic, grant number TN01000008, projects Instruct-Ultra (Grant 731005), iNEXT-Discovery (Grant 871037) funded by Horizon 2020 program of the European Commission, and the CIISB research infrastructure, an Instruct-ERIC Centre (LM2018127). We acknowledge the support obtained from Thermo Fisher Scientific Brno.
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