Name: advancing high-resolution imaging of virus assemblies in liquid and ice
Uploaded: 2022-07-20T13:30:00
Description: Watch this Scientific Journal Video about Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice at JoVE.com

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...

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>&#160;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>&#160;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.&#160;</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>Equipment&#160;</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>&#160;Pump holder tip to 10-6 range</td>
		</tr>
		<tr>
			<td>Mark IV Vitrobot</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>state-of-the-art specimen preparation unit&#160;</td>
		</tr>
		<tr>
			<td>PELCO easiGlow, glow discharge unit</td>
			<td>Ted Pella, Inc.</td>
			<td></td>
			<td>&#160;Negative polarity mode</td>
		</tr>
		<tr>
			<td>Poseidon Select specimen holder</td>
			<td>Protochips, Inc.</td>
			<td></td>
			<td>&#160;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>Freely available software</td>
			<td>Website link</td>
			<td></td>
			<td>Comments (optional)</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

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...

Watch this Scientific Journal Video about Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice at JoVE.com

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

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 ...

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Biochemistry

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

FÃƒÆ’Ã‚Â¶rster Resonance Energy Transfer Measurements in Living Plant Cells

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...