Name: Scanning Transmission Electron Microscopy Tomography in Virology: 3D Imaging of High-pressure Frozen, Freeze-substituted Samples (Video) | JoVE Journal
Uploaded: 2025-08-06T12:30:00.000+00:00
Duration: 9 min 17 s
Description: Learn STEM tomography for 3D imaging of virus-infected cells using high-pressure freezing and freeze substitution. This protocol enables comprehensive visualization of ultrastructural changes in thicker sections.

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This publication is part of the Global Development Technology Strategy and was supported by the Global Technology Management at Boehringer Ingelheim Development CMC Biologicals. We thank Renate Kunz and Jana Apolloni for excellent technical assistance and helpful discussion concerning sample preparation. We thank Reinhard Weih for excellent assistance with technical issues and concerning device maintenance. We thank Torsten Friedrich for helpful discussion and for aiding in the setup of the microscope. The work of Clarissa Read, Julia LaRoche, and Jan M&#252;nch has received funding by a Cooperative Research Centre Grant of the German Research Foundation (SFB1279).

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</ol>

JGW, RH, EP, JSR, and MD are employees of Boehringer Ingelheim Pharma GmbH &#38; Co.KG, a pharmaceutical company with an interest in developing and producing virus-based products. This had no influence on the design of the paper; therefore, we report no conflicts of interest.

STEM tomography offers a resolution comparable to TEM tomography combined with the ability to investigate volumes with up to 1 &#181;m thickness. We therefore consider it ideal for imaging virus-induced changes of intracellular structures, like membrane remodeling4,6,25, budding of virions7, or endocytosis5. Depending on the research question, other VolumeEM techniques might be considered. For a technique that provides a larger field of view with a comparable resolution, we recommend FIB-SEM tomography. FIB-SEM tomography will be slower than STEM tomography when imaging a comparable volume with similar resolution, since the imaging time increases when the resolution increases. Often, this is then countermeasured by imaging smaller fields of view. Furthermore, a z-resolution of 6.8 nm (Figure 6) would be technically challenging with ion beam milling. Therefore, FIB-SEM is optimal for imaging larger structures like whole cells and tissue9,10,26,27, albeit usually with (isotropic) pixel sizes of several tens of nanometers. Furthermore, FIB-SEM tomograms show no &#34;missing wedge&#34; effects, which is typical for tomograms reconstructed from a tilt series. Moreover, with the development of plasma FIB machines, alternatives that provide faster and still precise milling are currently being introduced to the field.
If a high resolution in z is not crucial, TEM or SEM imaging of serial sections can be an alternative that allows for the investigation of a very large field of view in a short time28,29, however, usually with anisotropic resolution. In TEM tomography, different tomograms from serial thick sections can be combined30,31. The same is possible in STEM tomography; however, given the thickness of the sections that can be investigated, there is less need to combine a number of tomograms. In virology, TEM tomography is often used to characterize virus-induced membrane remodeling32,33,34,35. While TEM tomography itself provides valuable insights in the replication cycle of viruses, most use-cases would benefit from the capability of STEM tomography to image thicker volumes. This especially holds true for virions with an anisotropic morphology, like rhabdoviridae, where TEM tomography can only capture a few complete virions in one tomogram35&#160;or for imaging several enveloping virions in a replication compartment with a size of the cytoplasmic viral assembly complex (cVAC) of the human cytomegalovirus (HCMV)4.
STEM tomography under cryogenic conditions offers further functional insights19 and a video protocol is available20. Our protocol differs in the sample preparation workflow, which, instead of preserving the sample in the native state, provides near-native ultrastructural preservation. While STEM tomography of high-pressure frozen, freeze-substituted samples cannot achieve the ultrastructural preservation and atomic resolution seen in cryogenic EM, it benefits from the advantages of simpler equipment, more stable specimens, and a higher throughput.
The following steps are critical for the success of the method:&#160;
First, a clear goal of the imaging experiment. Due to the labor-intensive nature of the technique, a well-planned experiment with a clear goal for imaging is needed. Detailed knowledge of the model system -- cell line and virus -- should be generated beforehand by using other methods that are less difficult to perform. Therefore, we encourage optimizing infectivity rates in light microscopy-based pre-experiments and adjusting experimental conditions to suit the biological system. Optimization parameters include the number of cells, multiplicity of infection (MOI), and time points based on the virus and the event to be imaged. This will ensure a high proportion of evaluable cells in the sample.
Second, high-quality sample preparation. We emphasize the importance of quality sample preparation. At the present time, we consider vitrification by high-pressure freezing as the gold standard for thick specimens like cells. Freeze substitution, embedding, and subsequent imaging at room temperature is a robust way of acquiring high-quality results when combined with STEM tomography. Therefore, establishing a stable routine for sample preparation is crucial. For this, we would like to highlight the literature about high-pressure freezing14,21. If a less toxic freeze-substitution solution is to be used, we recommend using potassium permanganate as a substitute for osmium tetroxide, as described by Schauflinger et al.36.
Finally, a smooth-running workflow for tilt series acquisition, including a well-aligned microscope, should be established in accordance with the user instructions of the STEM and will differ from microscope to microscope. Establishing a smooth workflow also depends on the scan generator and the image acquisition software.

<img alt="Cryo STEM tomography diagram comparing resolution, instrumentation, and effort in microscopy setups." class="xfigimg" src="/files/ftp_upload/68568/68568fig1v3.jpg" data-altupdatedat="1774324804000" data-alt="Figure 1"> 
Figure 1: Comparison of different 3D microscopy techniques, considering resolution, instrumentation, acquisition time, and effort. The techniques´ maximum feasible volume is between the technically demanding cryo-TEM tomography that offers molecular resolution, and light microscopy, where resolution is limited by the diffraction limit of light. Abbreviations: TEM = transmission electron microscopy; STEM = scanning TEM; SEM = scanning electron microscopy; FIB = focused ion beam. <a href="https://www.jove.com/files/ftp_upload/68568/68568fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Virus-related structures and systems span a scale from the sub-angstrom regime (e.g., small molecules for virotherapy, isolated viral proteins) to several meters (e.g., entire infected organisms). Viral particles are in the range of 20-1,000 nm and mainly investigated in their biological context, such as host cells and viral producer cell lines, which are in the µm range. Visualization of specific virus-related structures is thus dependent on choosing a technique that provides a sufficient field of view and suitable resolution. The study of virus-infected cells and tissue pieces relies on microscopy techniques (Figure 1). While overview images of cells and tissues can be acquired using classical light microscopy, intracellular structures involved in virus replication are often studied using electron microscopy (EM). Although nowadays mostly used in basic virological research, EM is also used in diagnostics1,2 and has also found its way into biopharmaceutical applications3.
Often, 2D EM approaches like classical transmission electron microscopy (TEM) cannot sufficiently answer a given biological question. In virology, this is especially the case when studying virus-host-cell interactions such as virion attachment and entry, the formation of viral factories, or virion morphogenesis4,5,6,7,8,9. Therefore, EM tomography is used to analyze the 3D ultrastructure of biological samples. In principle, there are two ways of acquiring a tomogram of a certain sample: 1) stepwise imaging of serial slices that are later combined into a 3D image stack, or 2) acquisition of projections from different viewing angles of the sample followed by computational reconstruction of the information into a virtual image stack.
The techniques of the first type include imaging of serial sections by TEM or SEM (SEM array tomography), serial block face SEM, and focused ion beam (FIB)-SEM tomography. SEM and TEM imaging of serial sections lack the resolution in z, which is limited by section thickness, but provides a large field of view and relies on equipment that is readily available in most EM labs4. FIB-SEM tomography can yield isotropic resolution, but device and maintenance costs can be challenging, and imaging of whole cells at isotropic resolution is time-consuming9,10.
The second type of tomography relies exclusively on the transmission of the electron beam through a sample. This can be done using two different imaging modalities: TEM or scanning transmission electron microscopy (STEM). While TEM tomography is more widespread and easier to implement, the section thickness is limited to ~200 nm. Therefore, structures larger than the section thickness, such as mitochondria, vesicles, or virions, cannot be imaged within a single tomogram. STEM tomography offers the possibility to image samples up to 1 µm thickness11,12. However, it requires a STEM with high acceleration voltage (≥200 kV) and a setup that supports high tilt angles as well as a small semi-convergence angle13. Although STEM imaging is a common technique in material science, it remains a rare technique in biologic EM laboratories.
Here, we present an application-driven approach to use STEM tomography for virological specimens. Due to the high complexity and labor-intensive nature of EM tomography, high-quality sample preparation is crucial. While cryogenic TEM (tomography) undoubtedly is the gold standard for investigations on a molecular level, room-temperature imaging of high-pressure frozen, freeze-substituted cells provides a solid preservation and clarity of cellular ultrastructure7,14. Briefly, adherent cells are grown on a sapphire disk and infected or otherwise treated as desired. The cells are then immobilized by freezing them under high pressure at very high cooling rates, resulting in vitrification of the water within. Subsequently, the water is replaced by a solvent while the samples are contrasted and slowly brought back to room temperature. The samples are then gradually embedded in resin.
There are many protocols for high-pressure freezing and freeze-substitution, which differ in the machinery, carrier system, and composition of the specimen. Detailed protocols for high-pressure freezing have been published, including one by Walther et al.15, and a video protocol by Steyer et al.16, which focuses on tissue preparation for FIB-SEM using microwave-assisted contrast enhancement. For virological research, Read et al.17 and Romero-Brey18 published detailed protocols for the preparation of virus-infected cells by high-pressure freezing with sapphire disks and subsequent analysis using electron tomography. Ultrathin sectioning has also been previously described15,17. Refer to the cited literature for more details regarding these approaches.
Here, we present a workflow that is an adapted and updated version of the previously published protocols15,17 paired for the first time with a detailed video guide. The protocol provides a workflow for sample preparation using high-pressure freezing, freeze substitution, and embedding in epoxy resin. Sections of 800 nm-1,000 nm thickness are cut using an ultramicrotome. A tilt series is acquired with a 200 kV STEM, using the software EM tools. This protocol is optimized for adherent cells and provides a comprehensive workflow while allowing modifications for other specimen types, such as tissue or suspension cells. For STEM tomography under cryogenic conditions, refer to the work of Michael Elbaum's group19,20.

<table><tbody><tr><td>1-Hexadecene</td><td>Merck KGaA</td><td>8220640500</td><td></td></tr><tr><td>200 copper grid with parallel bars</td><td>Plano GmbH</td><td>G2014C</td><td></td></tr><tr><td>37 &amp;deg;C drying chamber</td><td>Binder GmbH</td><td>9010-0323</td><td></td></tr><tr><td>Acetone</td><td>VWR international GmbH</td><td>20066330</td><td></td></tr><tr><td>DDSA</td><td>Electron Microscopy Sciences</td><td>13710</td><td></td></tr><tr><td>Diamond knife</td><td>Diatome Ltd</td><td>https://www.diatomeknives.com/ultra-35</td><td></td></tr><tr><td>DMP-30</td><td>Electron Microscopy Sciences</td><td>13600</td><td></td></tr><tr><td>Embed-812 Resin</td><td>Electron Microscopy Sciences</td><td>14900</td><td></td></tr><tr><td>EM tools</td><td>TVIPS GmbH</td><td>TVIPS Software: EM-Tools; https://www.tvips.com/imaging-software/em-tools/</td><td></td></tr><tr><td>Ethanol 100%</td><td>Merck KGaA</td><td>1.00983.2511</td><td></td></tr><tr><td>Filter paper</td><td>VWR international GmbH</td><td>516-0812</td><td></td></tr><tr><td>Flaschen mit Schnappdeckelglas 11 mL</td><td>VWR international GmbH</td><td>548-0625</td><td></td></tr><tr><td>Formvar</td><td>Electron Microscopy Sciences</td><td>15830-25</td><td></td></tr><tr><td>Freeze substitution unit Leica EM AFS2</td><td>Leica Microsystems GmbH</td><td>https://www.leica-microsystems.com/products/sample-preparation-for-electron-microscopy/p/leica-em-afs2/</td><td></td></tr><tr><td>Glod Sol 25 nm</td><td>AURION Immuno Gold Reagents &amp;amp; Accessories</td><td>425.011</td><td></td></tr><tr><td>Gold pinehole &amp;oslash; 2000 &amp;mu;m</td><td>Science Services GmbH</td><td>GA2000-Au</td><td></td></tr><tr><td>Graiticules Buchstaben-Netzchen Kupfer</td><td>Plano GmbH</td><td>NH6C</td><td></td></tr><tr><td>Heating plate</td><td>MEDAX GmbH &amp;amp; Co. KG</td><td>12501</td><td></td></tr><tr><td>High vacuum vaporization system BAF 300</td><td>BAL-TEC AG</td><td></td><td></td></tr><tr><td>High-pressure freezer Wohlwend HPF Compact 01</td><td>Engineering Office M. Wohlwend GmbH</td><td>https://www.wohlwend-hpf.ch/index.html</td><td></td></tr><tr><td>Liquid nitrogen tank</td><td>H.Erben GmbH</td><td>94500</td><td></td></tr><tr><td>Nitrogen (fluid)</td><td>VWR international GmbH</td><td>-</td><td></td></tr><tr><td>NMA</td><td>Electron Microscopy Sciences</td><td>19000</td><td></td></tr><tr><td>Osmium tetroxide</td><td>ChemPur Feinchemikalien und Forschungsbedarf GmbH</td><td>006051</td><td></td></tr><tr><td>Oven 120 &amp;deg;C</td><td>Binder GmbH</td><td>9010-0194</td><td></td></tr><tr><td>PELCO easi-Glow</td><td>Ted Pella, Inc.</td><td>91000</td><td></td></tr><tr><td>Poly-L-Lysin 0.1% w/v aq. Solution</td><td>Ted Pella, Inc.</td><td>18026</td><td></td></tr><tr><td>Safe-Lock Tubes 0.5 mL</td><td>Eppendorf SE</td><td>0030 121.023</td><td></td></tr><tr><td>Sample containers (Beem capsules)</td><td>Plano GmbH</td><td>G360-1</td><td></td></tr><tr><td>Sapphire disc dia. 3 x 0.16 mm</td><td>Engineering Office M. Wohlwend GmbH</td><td>500</td><td></td></tr><tr><td>Ultramicrotome Leica EM UC7</td><td>Leica Microsystems GmbH</td><td>https://www.leica-microsystems.com/de/produkte/em-probenvorbereitung/p/leica-em-uc7/</td><td></td></tr><tr><td>Uranyl acetate</td><td>Riedel de Ha&amp;euml;n AG</td><td>31697</td><td></td></tr><tr><td>UV light chamber</td><td>Dinies</td><td>ELG100S</td><td></td></tr></tbody></table>

scanning transmission electron microscopy tomography in virology: 3d imaging of high-pressure frozen, freeze-substituted samples

Scanning Transmission Electron Microscopy Tomography in Virology: 3D Imaging of High-pressure Frozen, Freeze-substituted Samples

Electron microscopy (EM) and especially three-dimensional (3D) EM techniques have become established methods in structural virology. Investigation of virus-induced alteration of the cellular ultrastructure, such as Zika virus (ZIKV)-induced replication factories or coronavirus replication organelles, demands 3D imaging. Transmission electron microscopy (TEM) tomography is a widely used method, despite its limitation to samples with a thickness of up to 200 nm. Focused ion beam-scanning electron microscopy (SEM)-tomography can produce 3D data of larger volumes with isotropic, albeit typically lower resolution. Alternative techniques, such as block face-scanning electron microscopy (BF-SEM), SEM array tomography, or TEM imaging of serial sections are used for imaging of larger volumes. However, compared to the previously mentioned techniques, these techniques come at the cost of much lower resolution along the Z-axis of the sample. A technique that provides 3D information of samples up to 1 &#181;m thickness with isotropic resolution of a few nanometers is scanning transmission electron microscopy (STEM) tomography.
Here, we present a protocol for the preparation of high-pressure frozen, freeze-substituted, and resin-embedded virological specimens and their analysis using STEM tomography. This protocol benefits from the advantages of room temperature imaging while preserving the biological ultrastructure in a near-native state. We show two representative examples for questions that can be answered using STEM tomography. First, we apply the protocol for studying virion morphogenesis of a recombinant vesicular stomatitis virus (VSV). The STEM tomograms offer information on recombinant VSV budding that is otherwise not accessible by 2D imaging. Second, we show correlative light and electron microscopy (CLEM) using Foerster resonance energy transfer (FRET) imaging and STEM tomography (FRET-3D-CLEM) of EF-C peptide nanofibrils. This recently published combination gives new insights into the uptake and disassembly of infection-enhancing peptide nanofibrils, especially profiting from the large volume that is accessible by STEM tomography.

STEM tomography offers the possibility to image thicker sections than TEM tomography with isotropic resolution in the nanometer range and-as compared to FIB-SEM tomography-provides a relatively time-efficient imaging workflow. In virology, this is especially useful since it allows for imaging of a large number of ultrastructural features for statistical analysis in 3D, or when searching for rare events. Here, we show two representative results that demonstrate how STEM tomography can be used with regard to these two aspects.
Visualization of budding recombinant VSV virions
There is a broad variety of viruses that form particles of different shapes and sizes. To analyze the shape of virions in a virus suspension, often a simple method like negative stain TEM is sufficient. Characterizing the morphology of virions in the cellular context is more difficult. This holds especially true for viruses that form particles with anisotropic morphology, like rhabdoviridae. VSV is a rhabdovirus that forms bullet-shaped virions of ~180 nm in length. In suspension cells, the virions are randomly oriented, making it difficult to always discern the bullet shape in 2D images (Figure 5A-C). To properly define the bullet shape of all recombinant VSV virions in a sample, a 3D method with isotropic resolution is needed-one that can also provide volumetric information over a sufficiently large sample area. A STEM tomogram can include many complete virions in a single dataset (Figure 5D,E). Thereby, it is possible to image a high number of virions for statistical analysis, even if only one tomogram per cell is acquired. In comparison, a TEM tomogram of a 200 nm-thick section would only capture a few complete recombinant VSV virions (as marked in Figure 5E).
<img alt="Cryo-EM micrograph of protein structure, structural analysis at nanoscale, labeled sections A-E." class="xfigimg" src="/files/ftp_upload/68568/68568fig5.jpg" data-altupdatedat="1774324804000" data-alt="Figure 5"> 
Figure 5: STEM tomography of recombinant VSV budding sites. (A-C) Details from a virtual section of a STEM tomogram obtained from an 800 nm thick section show that the bullet shape is not always visible due to (A,B) different orientations of the virions. (C) Only virions that are oriented parallel to the viewing plane present as well-defined bullets. (D) Budding sites of recombinant VSV resemble large clusters of virions with no preferred orientation. (E) The side view of the tomogram demonstrates the ability to capture many complete recombinant VSV virions in one section using STEM tomography. The virtual image stack has a thickness of approximately 600 nm at this position. The dashed lines represent the approximate thickness of a section for TEM tomography (200 nm), where only a few complete bullet-shaped virions would be included. Scale bar = 500 nm (D). Abbreviations: STEM = scanning transmission electron microscopy; VSV = vesicular stomatitis virus. <a href="https://www.jove.com/files/ftp_upload/68568/68568fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Degradation of EF-C peptide nanofibrils
In the context of gene therapy, transduction-enhancing peptide nanofibrils have become a topic of interest. In a recently published study24, correlative imaging using Foerster Resonance Energy Transfer (FRET) imaging and STEM tomography (FRET-3D-CLEM) was used to investigate the uptake and disassembly of EF-C peptide nanofibrils. High FRET efficiencies, corresponding to FRET pairs that are close to each other, emit red fluorescence corresponding to assembled fibrils. Low FRET efficiencies, corresponding to FRET pairs that are far from each other, emit green fluorescence, representing disassembled fibrils. Both signals appear in cells when they are in the process of taking up and degrading EF-C peptide nanofibrils. Areas where high and low FRET efficiencies are found appear in yellow (Figure 6A).
<img alt="Cellular ultrastructure analysis, electron microscopy images and segmentation results, highlighting nucleolus." class="xfigimg" src="/files/ftp_upload/68568/68568fig6v2.jpg" data-altupdatedat="1774324804000" data-alt="Figure 6"> 
Figure 6: Imaging of EF-C peptide nanofibril degradation by correlative FRET imaging and STEM tomography (FRET-3D-CLEM). (A) Confocal microscopy of a cell incubated with EF-C nanofibrils that were labeled with a FRET pair. Green signals correspond to putatively disassembled fibrils. Red corresponds to putatively intact fibrils. Yellow corresponds to areas where a mixture of the red and the green signal is detected. Nu nucleus. (B) STEM overview of an 800 nm-thick section from the same cell. (C) The position of the tomogram is indicated. The image is a composite of two images acquired on different sections, because the cell was partially blocked by the bar grid on one of the sections. The nucleus is labeled with Nu in A, B, and C. (C) Virtual section from the tomogram. Red arrowheads mark regions overlapping with red fluorescence signals. STEM tomography confirms the presence of intact fibrils. The green arrowhead points to a region with disassembled fibrils. (D) Segmentation of the tomogram. An endocytosed filopodium is pinching off the vesicle membrane (white arrow). The large volume that can be investigated with a STEM tomogram (here approximately: 7 µm x 7 µm x 550 nm) makes it easier to observe such a rare event. (E) Virtual sections 19, 24, and 29 of the tomogram. At a pixel size of 6.8 nm, the sections are each 27.2 nm apart. The figure is reproduced from the data published by Rauch-Wirth et al.24 . Scale bars = 5 µm (A), 1 µm (C). Abbreviations: STEM = scanning transmission electron microscopy; FRET = Foerster resonance energy transfer. <a href="https://www.jove.com/files/ftp_upload/68568/68568fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Visualization was performed by this correlative approach to confirm that EF-C fibrils are taken up by the cell and subsequently degraded. Light microscopy lets us choose cells for electron microscopy that provide red and green signals, to compare the ultrastructural appearance correlating with the different fluorescence signals. STEM tomograms from the same cell show that the red signal can either originate from fibrils that are still outside the cell or are seemingly freshly internalized by macropinocytosis (Figure 6C). Furthermore, CLEM verified that the green signal indeed originated from degraded fibrils (Figure 6B,C which can be found in lysosomes (Figure 6C, largest vesicle). Due to the large volume accessible by STEM tomography and the 3D visualization of the structures, it is easier to correlate the signal from light microscopy with ultrastructural features and interpret them. Furthermore, rare events, such as pinching-off filopodia inside the lysosome (Figure 6D,E, white arrow) can be detected with this approach.

This video protocol illustrates how to perform scanning transmission electron microscopy tomography of virological specimens. For optimal outcomes, samples are prepared by high-pressure freezing and subsequent freeze substitution.

Electron microscopy (EM) and especially three-dimensional (3D) EM techniques have become established methods in structural virology. Investigation of ...

Viral infection could cause significant alterations in cellular infrastructure. To visualize these changes in three dimensions, we employ STEM tomography for imaging of virus infected cells. STEM tomography is time-consuming and labor-intensive.This makes optimal sample preparation essential for biologic specimens, such as virus-infected sets. Conventional STEM tomography is limited to sample sicknesses of approximately 200 nanometers. Our protocol enables 3D imaging of thicker sections.This allows a more comprehensive visualization of virus infected cells and the ultra structural alterations. Many oncolytic viruses are originally clinical trials already, therefore, researcher and developer worldwide working together, and the science is pushing the border of cancer treatment and state-of-the-art methods, like electron microscopy and STEM tomography contribute with new insights. We'll continue to answer virological questions by using STEM tomography and combining it with new preparation protocols and other microscopy techniques.To begin, fill the LN-box with liquid nitrogen in preparation for handling and transferring the frozen sapphire discs from the high pressure freezing or HPF holder to the storage capsules. Keep a dewar filled with liquid nitrogen nearby to refill the LN-box as needed during the freezing process. Keep a pair of insulated tweezers ready.Prepare the sample containers that will hold the sapphire discs after HPF. Make sure that they have a lid and several holes for liquid nitrogen to pour in and small weights to ensure that they do not float. Place the label container in the LN-box to pre-cool before using.Then, place the samples in the incubator. Inspect the sapphire discs under an inverted light microscope to ensure enough cells and good cell quality before starting high pressure freezing. To perform HPF, take one sapphire disc from the culture dish and quickly dab it on filter paper to remove excess media.Place the disc in the HPF holder with the carbon-coated side facing upwards, ensuring the number two is readable. Dip a gold spacer into hexadecane and place it gently on the sapphire disc. Position a second sapphire disc on top of the spacer with the carbon coated side facing downwards to form a sandwich configuration with the cells pointing towards each other, ensure there is no air trapped.Close the HPF holder carefully and quickly to reduce stress on the sample. Insert the HPF holder into the high pressure freezer. Lock it according to the manufacturer's instructions and initiate the freezing process.Next, transfer the HPF holder to the LN-box. Then transfer the tip carrying the sapphire discs into the pre-cool LN-box. Carefully extract the discs from the holder.Place the closed cryo vials filled with free substitution solution into a free substitution device pre-cooled to minus 90 degrees Celsius. Wait until the solution reaches the same temperature. Pre-cool tweezers, a scalpel and pliers or large tweezers by immersing them in liquid nitrogen for use in transferring the sapphire discs.Transfer the high pressure frozen samples into the free substitution device. Separate the sapphire discs one by one, while keeping them in liquid nitrogen. And quickly transfer each disc individually into a cryo vial with free substitution solution, ensuring that the disc is fully immersed.After closing the cryo vial, immediately place it back into the free substitution device. Once all samples are placed, start the substitution process. As soon as the samples reach 20 degrees Celsius.Continue with washing the samples three times for 30 minutes each with 100%acetone. To pre-embed the samples, incubate the samples in one third epoxy resin and two thirds acetone for one hour. After incubating the samples with two thirds epoxy resin and one third acetone for one hour, exchange the solution with 100%epoxy resin and incubate overnight with the vial lid open to allow remaining acetone to evaporate on the following day, transfer the sapphire discs into new labeled reaction tubes filled with freshly prepared epoxy resin.Position the discs horizontally supported by the wall of the tube, ensuring that the side with cells is facing upward. Then place the reaction tubes with the lids open in an oven set to 60 degrees Celsius and allow the resin to polymerize for at least 48 hours. After the epoxy resin has polymerized, immerse the tip of a closed reaction tube in liquid nitrogen for approximately 10 seconds.Hit the reaction tube on a table to release the resin block. The sapphire disc will separate easily from the carbon coat, which remains attached to the embedded cells. Select the region of interest in the embedded sample and narrow down the sample size to the region of interest.Using an ultra microtone with a diamond knife, collect sections of around one micrometer thickness on a Poly-L-lysine treated glotus charged parallel bar grid for STEM imaging and STEM tomography. Treat sections with colloidal gold as fiducial markers and apply a carbon coating. After cutting and inspecting ultra thin sections, the samples are ready for imaging.Based on the research question, insert either the bright field detector, the dark field detector, or both. Activate the scan mode and acquire a test image of the sample. Select a magnification higher than 800, 000 times, which is larger than the final desired magnification.Near the region of interest, adjust the focus and correct astigmatism while avoiding direct beam exposure on the critical sample areas. Set the eccentric height so the region of interest, or ROI stays centered throughout the tilt range. Choose a feature near the ROI bring it to the center, focus, correct astigmatism, and tilt the stage stepwise to minus 72 degrees.To choose the field of view, navigate back to the ROI bring it into focus and capture an overview image. Set the final magnification desired for tilt series acquisition. Set up automatic tilt series acquisition of images, ranging from minus 72 degrees to plus 72 degrees and image every 1.5 degrees.Start the automated tilt series acquisition. Representative virtual slices from the STEM thermogram illustrated how virions orientation impacts the visibility of the characteristic bullet shape in two-dimensional views. Only virions aligned parallel to the viewing plane appeared with the full well-defined bullet morphology, while others appeared circular or elongated, depending on their tilt angle.To overcome the orientation limitations of 2D imaging, STEM tomography was used to capture large clusters of budding recombinant vesicular stomatitis virus virion enabling assessment of virion shape in three dimensions without directional bias. The side view of thermogram confirmed the volumetric advantage of STEM tomography capturing many complete virions within a single 600-nanometer thick section, in contrast to the limited sampling possible with a 200-nanometer TEM section.

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JoVE Journal

Medicine

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing.  It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein.  These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data...

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

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Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

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Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

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Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

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A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

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.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Cryo-STEM tomography provides a means to visualize organelles of intact cells without embedding, sectioning, or other invasive preparations. The 3D resolution obtained is currently in the range of a few nanometers, with a field of view of several micrometers and an accessible thickness in the order of 1 µm.

Visualization of Organelles In Situ by Cryo-STEM Tomography

Learn STEM tomography for 3D imaging of virus-infected cells using high-pressure freezing and freeze substitution. This protocol enables comprehensive visualization of ultrastructural changes in thicker sections.

Scanning Transmission Electron Microscopy Tomography in Virology: 3D Imaging of High-pressure Frozen, Freeze-substituted Samples (Video) | JoVE Journal

Scanning Transmission Electron Microscopy Tomography in Virology: 3D Imaging of High-pressure Frozen, Freeze-substituted Samples

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