Name: Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice (Video) | JoVE Journal
Uploaded: 2022-07-20T13:30:00.000+00:00
Duration: 8 min 31 s
Description: Learn liquid-EM and cryo-EM protocols for high-resolution imaging of virus assemblies at the nanoscale. These methods enable dynamic structural analysis of pathogens like SARS-CoV-2 using transmission electron microscopy.

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5 minutes

The authors acknowledge Dr. Luk H. Vandenberghe (Harvard Medical School, Department of Ophthalmology) for providing purified AAV-3. This work was supported by the National Institutes of Health and the National Cancer Institute (R01CA193578, R01CA227261, R01CA219700 to D.F.K.).

1. Loading the specimen holder for liquid-EM
<ol>
	<li>Clean the silicon nitride (SiN) microchips by incubating each chip in 150 mL of acetone for 2 min followed by incubation in 150 mL of methanol for 2 min. Allow chips to dry in laminar airflow.</li>
	<li>Plasma clean the dried chips using a glow-discharge instrument operating under standard conditions of 30 W, 15 mA for 45 s using Argon gas.</li>
	<li>Load a dry base microchip into the tip of the specimen holder. Add ~0.2 &#181;L of sample (0.2-1 mg/mL of virus assemblies in 50 mM HEPES, pH 7.5; 150 mM NaCl; 10 mM MgCl2; 10 mM CaCl2) to the base chip. Following a 1-2 min incubation step, place the top chip on the wet base chip containing the sample.</li>
	<li>Clamp the assembly together to form a hermetically sealed enclosure, held in place mechanically by three brass screws. Upon sealing the assembly, pump the tip to 10-6 Torr using a turbo-pumped dry pumping station. The holder is now ready to be inserted into the TEM. 
	NOTE: The select holder has no cooling capabilities and is not used for cryo-EM.</li>
</ol>
2. Production of microchip sandwich assemblies
NOTE: Different SiN or silicon dioxide (SiO) microchips can be used directly from the shipped gel packs. Carbon-coated gold grids may also be used directly as supplied.
<ol>
	<li>Plasma clean the microchips and carbon grids using a glow discharge instrument operating under standard conditions of 30 W, 15 mA for 45 s using Argon gas.</li>
	<li>Add ~2 &#181;L of sample contained in 50 mM HEPES, pH 7.5; 150 mM NaCl; 10 mM MgCl2; 10 mM CaCl2&#160;to a glow-discharged microchip placed on a gel pack. Remove excess solution (~50%) using a filter paper or a pipette. Following a 1-2 min incubation step, add the glow-discharged carbon grid to the wet microchip containing the sample.</li>
	<li>Clamp the assembly together using a single-tilt specimen holder or autoloader grid clips at room temperature to form a hermetically sealed enclosure. For the auto-loader clamps, place the sandwich assembly on the bottom C-clip, place the top clip on top of the assembly and use the standard clamping tool to seal the assembly together. 
	NOTE: The clamping procedure performed here uses the same steps as for cryo-EM grid clamping, but at room temperature. Clamped samples may be stored for 2 months or longer prior to imaging while maintaining liquid in the enclosure. No leak check is used with a room temperature specimen holder or the autoloader.</li>
	<li>The specimen is now ready to be inserted into the TEM. Examine samples placed in autoloaders under cryo conditions or at room temperature.</li>
</ol>
3. Imaging specimens using a transmission electron microscope
<ol>
	<li>Liquid-EM imaging
	<ol>
		<li>Load the&#160;specimen holder into the TEM equipped with a field-emission gun (FEG) and operating at 200 kV.</li>
		<li>Turn on the gun and adjust the eucentric height of the microscope stage with respect to the specimen by using the wobbler function, tilting the sample from -15&#176; to +15&#176; in the column. This procedure adjusts the stage in the Z-direction to accommodate the sample thickness. And helps ensure an accurate magnification during image recording.</li>
		<li>Record images as long-framed movies using the in situ package integrated with a direct detector having a pixel spacing of 6 &#181;m (Video 1&#160;and Video 2). Individual images may also be recorded using the serial data collection software package20, implementing automated imaging routines. Acquire images under low-dose conditions at magnifications ranging from 28,000x-92,000x and 40 frames per second.</li>
		<li>Adjust exposure times (0.25-1 s) to minimize beam damage to the specimen. Use a defocus range of -1-4 &#181;m at the specified magnification. If a thick solution is encountered, use higher defocus values or select a different region of interest.</li>
		<li>Ensure the solution is present in the samples throughout the imaging session by focusing the electron beam on a sacrificial area not used for data collection, until bubbles are formed (Supplementary Figure 1).</li>
	</ol>
	</li>
	<li>Cryo-EM imaging
	<ol>
		<li>Load the&#160;clipped EM grids or microchip sandwiches into the TEM equipped with a FEG and operating at 300 kV. Turn on the gun and adjust the eucentric height of the microscope stage, using a similar procedure described for the liquid-TEM above (step 3.1.2).</li>
		<li>Record individual images using the single particle analysis system integrated within the microscope system while implementing automated imaging routines. Record images under low-dose conditions using the single particle analysis direct electron detector having a pixel spacing of 14 &#181;m at a magnification of 59,000x and 40 frames per second.</li>
		<li>Use a defocus range of 1-4 &#181;m at the specified magnification. If thick layers of vitreous ice are encountered, use higher defocus values, or select a different region for data collection.</li>
	</ol>
	</li>
</ol>
4. Data analysis and 3 D structure comparisons
<ol>
	<li>Analysis of adeno-associated virus (AAV) in liquid and vitreous ice
	<ol>
		<li>Process movies for AAV particles in liquid and ice using RELION-3.08 program21 or other image processing software22. Perform motion correction using MotionCor2 v1.2.3.</li>
		<li>Once corrected, extract particles using the auto-picking tool in the program software package. Typical box sizes are 330 pixels for liquid specimens and 350 pixels for ice specimens.</li>
		<li>Calculate initial reconstructions using C1 symmetry using the program&#39;s 3D initial model routine and/or the ab-initio model options in the data processing software package. In the program, use a regularization parameter of T = 4 and a pixel size of 1.01 A&#778; for liquid specimens and 1.13 A&#778; for ice specimens.</li>
		<li>Use a mask value of 300 A&#778; throughout the refinement procedures. Perform refinement protocols in the data processing software using I1 symmetry to obtain multiple EM maps. Expected structural resolution may be in the range of 4 A&#778; or better. Use particle equivalency implementing icosahedral symmetry at 16,800 for liquid and 15,240 for ice12,14. 
		NOTE: Resolution estimates are based on the gold-standard Fourier shell correlation (FSC) criteria.</li>
		<li>Mask EM maps at ~250 A&#778; and examine results using a molecular structure analysis software package23,24. In Figure 3, slices are shown in increments of ~5 nm.</li>
		<li>Extract capsid protein (VP1) subunits from the EM maps for comparison. Quantify dynamic changes among EM structures based on the particle diameter measurements (Supplementary Figure 2), and then visualize using the Morph map function in the software.</li>
	</ol>
	</li>
	<li>Analysis of SARS-CoV-2 sub-viral assemblies in liquid
	<ol>
		<li>Process movies using the RELION-3.08 program. Correct for image drift and beam-induced motion using MotionCor2 v1.2.3. Correct for the contrast transfer function (CTF) of the microscope.</li>
		<li>Use the auto-picking tool in the program to select viral assemblies with a box size of 800 pixels. For computational efficiency, extracted particles can be rescaled to 256 pixels. Obtain an initial model using C1 symmetry in the program with a regularization parameter of T = 2&#160;and 1.66 A&#778; pixel size.</li>
		<li>Perform 3D refinement in the program to obtain an EM map, an example is shown at ~8.25 A&#778; according to standard FSC criteria (Figure 4). Visualize EM maps using the molecular structure analysis software with slices incremented at 25 nm as demonstrated in Figure 4.</li>
	</ol>
	</li>
	<li>Analysis of rotavirus double-layered particles (DLPs) in vitreous ice
	<ol>
		<li>Process movies for rotavirus DLPs using other image processing software. Use MotionCor2 v1.2.3 to correct for drift in the images. Use Patch CTF estimation to correct for lens effects on the images.</li>
		<li>Extract particles using the auto-picking tool in the software with a box size of 950 pixels. Down-sample the box size as needed for computing purposes.</li>
		<li>Calculate initial models using ab-initio options and C1 symmetry. Use refinement parameters including a pixel size of 1.47 A&#778; and a mask value of 800 A&#778;.</li>
		<li>Perform additional refinement routines while imposing I1 symmetry. Example results include a 10.15 A&#778; EM map, according to the gold-standard FSC criteria. Total particles used were 2050, which equates to 123,000 protomer units due to the icosahedral symmetry operator (Figure 5).</li>
		<li>Mask final maps at ~750 A&#778; and visualize results in the molecular structure analysis software package. Example slices through the EM map are shown in Figure 5 in increments of ~10 nm.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors declare that they have no competing financial interests. The author, Madeline J. Dressel-Dukes, is an employee of Protochips, Inc. and Michael Spilman is an employee of DirectElectron.

New opportunities are presented to streamline current liquid-EM workflows by using new automated tools and technologies adapted from the cryo-EM field. Applications involving the new microchip sandwich technique are significant with respect to other methods because they enable high-resolution imaging analysis in liquid or vitreous ice. One of the most critical steps in the protocol is producing specimens with the ideal liquid thickness to visualize exquisite details at the nanoscale level. Ideal regions of interest are i...

Biomedical research improves our understanding of human health and disease through the development of new technologies. High-resolution imaging is transforming our view of the nanoworld - permitting us to study cells and molecules in exquisite detail1,2,3,4,5. Static information of dynamic components such as soft polymers, protein assemblies, or human viruses reveals only a limited snapshot of their complex narrative. To better understand how molecular entities operate, their structure and function must be jointly investigated.
Recent advances in the production of materials such as atomically thin graphene&#160;or silicon-based microchips provide new opportunities for real-time structure-function analysis using transmission electron microscopes (TEMs). These materials can create hermetically sealed chambers for live EM imaging6,7,8,9,10,11. The new field of liquid-EM, the room temperature correlate to cryo-EM, provides unprecedented views of hard or soft materials in solution, allowing scientists to simultaneously study the structure and dynamics of their specimen. Liquid-EM applications include real-time recordings of therapeutic nanoparticles interacting with cancer stem cells as well as changes in the molecular intricacies of viral pathogens12,13,14.
Just as methodological advances spurred the resolution revolution in the cryo-EM field, new techniques and methods are needed to extend the use of liquid-EM as a high-throughput tool for the scientific community. The overall goal of the methods presented here is to streamline liquid-EM specimen preparation protocols. The rationale behind the developed techniques is to employ new microchip designs and autoloader devices, suitable for both liquid- and cryo-EM data collection (Figure 1)7,14,15,16,17. The assemblies are mechanically sealed using standard grid clips for automated instruments, such as the Krios, which can accommodate multiple samples per session or a F200C TEM (Figure 2). This methodology expands the use of high-resolution imaging beyond standard cryo-EM applications demonstrating broader purposes for real-time materials analysis.
In the current video article, protocols are presented for preparing virus assemblies in liquid with and without commercially available specimen holders. Using the specialized specimen holder for liquid-EM, thin liquid specimens can provide structural information comparable to cryo-EM samples, as well as dynamic insights of the specimens. Also demonstrated are methods for preparing liquid specimens using autoloader tools for high-throughput routines. The major advantage over other techniques is that automated specimen production allows the user to quickly assess their samples for optimum thickness and electron dosage prior to data collection. This screening technique quickly identifies ideal areas for real-time recordings in liquid or ice12,14,18,19. For purposes of 3D structure determination, liquid-EM may complement the long-established cryo-EM methods implemented in cryo-EM. Readers employing conventional TEM or cryo-EM technologies may consider using liquid-EM workflows to provide new, dynamic observations of their samples in a manner that complements their current strategies.
Virus samples used in this protocol include purified adeno-associated virus subtype 3 (AAV) obtained as a gift and cultured under standard conditions12. Also used were non-infectious SARS CoV-2 sub-viral assemblies derived from the serum of COVID-19 patients12&#160;and obtained from a commercial source. Finally, purified simian rotavirus (SA11 strain) double-layered particles (DLPs) were obtained from the laboratory of Dr. Sarah M. McDonald Esstman at Wake Forest University and cultured using standard conditions 6,17. Software packages described here are freely available and the links have been provided in the Table of Materials section.

<table><tbody><tr><td>Acetone</td><td>Fisher Scientific</td><td>&amp;nbsp;A11-1</td><td>1 Liter</td></tr><tr><td>Autoloader clipping tool</td><td>ThermoFisher Scientific</td><td>N/A</td><td>Also SubAngstrom supplier</td></tr><tr><td>Autoloader grid clips</td><td>ThermoFisher Scientific</td><td>N/A</td><td>top and bottom clips</td></tr><tr><td>Carbon-coated gold EM grids</td><td>Electron Microcopy Sciences</td><td>CF400-AU-50</td><td>400-mesh, 5-nm thickness</td></tr><tr><td>COVID-19 patient serum</td><td>RayBiotech</td><td>CoV-Pos-S-500</td><td>500 microliters of PCR+ serum</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>&amp;nbsp;A412-1</td><td>1 Liter</td></tr><tr><td>Microwell-integrad microchips</td><td>Protochips, Inc.</td><td>EPB-42A1-10</td><td>10x10-mm window arrays</td></tr><tr><td>TEMWindows microchips</td><td>Simpore Inc.</td><td>SN100-A10Q33B</td><td>9 large windows, 10-nn thick</td></tr><tr><td>TEMWindows microchips</td><td>Simpore, Inc.&amp;nbsp;</td><td>SN100-A05Q33A</td><td>9 small windows, 5-nm thick</td></tr><tr><td>Top microchips</td><td>Protochips, Inc.</td><td>EPT-50W</td><td>500 mm x 100 mm window</td></tr><tr><td>Whatman #1 filter paper</td><td>Whatman</td><td>1001 090</td><td>100 pieces, 90 mm</td></tr><tr><td>&lt;strong&gt;Equipment&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DirectView direct electron detector</td><td>Direct Electron</td><td></td><td>6-micron pixel spacing</td></tr><tr><td>Falcon 3 EC direct electron detector</td><td>ThermoFisher Scientific</td><td></td><td>14-micron pixel spacing</td></tr><tr><td>Gatan 655 Dry pump station</td><td>Gatan, Inc.</td><td></td><td>&amp;nbsp;Pump holder tip to 10&lt;sup&gt;-6 &lt;/sup&gt;range</td></tr><tr><td>Mark IV Vitrobot</td><td>ThermoFisher Scientific</td><td></td><td>state-of-the-art specimen preparation unit&amp;nbsp;</td></tr><tr><td>PELCO easiGlow, glow discharge unit</td><td>Ted Pella, Inc.</td><td></td><td>&amp;nbsp;Negative polarity mode</td></tr><tr><td>Poseidon Select specimen holder</td><td>Protochips, Inc.</td><td></td><td>&amp;nbsp;FEI compatible;specimen holder</td></tr><tr><td>Talos F200C TEM</td><td>ThermoFisher Scientific</td><td></td><td>200 kV; Liquid-TEM</td></tr><tr><td>Titan Krios G3</td><td>ThermoFisher Scientific</td><td></td><td>300 kV; Cryo-TEM</td></tr><tr><td>&lt;strong&gt;Freely available software&lt;/strong&gt;</td><td>&lt;strong&gt;Website link&lt;/strong&gt;</td><td></td><td>&lt;strong&gt;Comments (optional)&lt;/strong&gt;</td></tr><tr><td>cryoSPARC</td><td>https://cryosparc.com/</td><td></td><td>other image processing software</td></tr><tr><td>CTFFIND4</td><td>https://grigoriefflab.umassmed.edu/ctffind4</td><td></td><td>CTF finding program</td></tr><tr><td>MotionCorr2</td><td>https://emcore.ucsf.edu/ucsf-software</td><td></td><td></td></tr><tr><td>RELION</td><td>https://www3.mrc-lmb.cam.ac.uk/relion/index.php?title=Main_Page</td><td></td><td></td></tr><tr><td>SerialEM</td><td>https://bio3d.colorado.edu/SerialEM/</td><td></td><td></td></tr><tr><td>UCSF Chimera</td><td>https://www.cgl.ucsf.edu/chimera/</td><td></td><td>molecular structure analysis software package</td></tr></tbody></table>

advancing high-resolution imaging of virus assemblies in liquid and ice

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

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

A liquid-TEM operating at 200 kV was used for all liquid-EM imaging experiments and a cryo-TEM operating at 300 kV was used for all cryo-EM data collection. Representative images and structures of multiple viruses are presented to demonstrate the utility of the methods across various test subjects. These include recombinant adeno-associated virus subtype 3 (AAV), SARS-CoV-2 sub-viral assemblies derived from the patient serum, and simian rotavirus double-layered particles (DLPs), SA11 strain. First, comparisons are demons...

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

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

advancing-high-resolution-imaging-virus-assemblies-liquid

Research

JoVE Journal

Biochemistry

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

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

In situ TEM of Biological Assemblies in Liquid

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

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

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Single Particle Cryo-Electron Microscopy: From Sample to Structure

A newly developed micro-patterned chip with graphene oxide windows is fabricated by applying microelectromechanical system techniques, enabling efficient and high-throughput cryogenic electron microscopy imaging of various biomolecules and nanomaterials.

Fabrication of Micro-Patterned Chip with Controlled Thickness for High-Throughput Cryogenic Electron Microscopy

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.

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

This article presents a protocol for data processing of influenza viruses imaged using cryo-electron tomography and subsequent subtomogram averaging of the hemagglutinin glycoprotein. This protocol covers step-by-step data processing, from image preprocessing to final model refinement.

Subnanometer-Resolution Structural Determination of Hemagglutinin from Cryo-Electron Tomography of Influenza Viruses

Learn liquid-EM and cryo-EM protocols for high-resolution imaging of virus assemblies at the nanoscale. These methods enable dynamic structural analysis of pathogens like SARS-CoV-2 using transmission electron microscopy.

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

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