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 ...

Interest in liquid electron microscopy has skyrocketed in recent years as we can now visualize at the nanoscale realtime processes. Improved knowledge on these flexible structures can assist us in developing novel reagents to combat emerging pathogens such as SARS-CoV-2. Viewing proteins in a fluid environment helps to mimic biological systems and gives us an opportunity to look at the proteins in a more dynamic way.And this experiment will show you new techniques on how to use and visualize proteins in both vitreous ice and liquid environments. Recent results include dynamic insights of vaccine candidates in antibody-based therapies imaged in liquid. Correlative liquid and cryo-EM applications advance our ability to visualize molecular dynamics, providing a unique context for human health and disease.Readers employing conventional TEM or cryo-EM technologies may consider implementing liquid EM workflows to provide new dynamic observations of their samples in a way that complements their current strategies. Commercially available liquid-EM systems can provide flow, mixing, electrochemical stimuli, and temperature control, which are essential for many realtime imaging applications. The microchip sandwich method presented here describes a simple entry level way to first view specimens in liquid before making the leap to a more complex commercial system for in situ experiments.To begin, clean the silicon nitride microchips by incubating each chip in 150 milliliters of acetone for two minutes, followed by incubation in 150 milliliters of methanol for two minutes. Allow chips to dry in a laminar airflow. Plasma clean the dried chips using a glow discharge instrument operating under standard conditions for 45 seconds using argon gas.Next, load a dry base microchip into the tip of the specimen holder and add around 0.2 microliters of the sample to the base chip. After incubating for one to two minutes, place the top chip on the wet base chip containing the sample. Plant the assembly together to form a hermetically sealed enclosure held in place mechanically by three brass screws.Pump the tip to 10 to the minus six torr using a turbo pumped dry pumping station. The holder is now ready to be inserted into the TEM. Plasma clean the microchips and carbon grids using a glow discharge instrument for 45 seconds.And add around two microliters of sample to a glow discharged microchip placed on a gel pack. Remove the excess solution using filter paper or a pipette and incubate for one to two minutes. Then add the glow discharged carbon grid to the wet microchip containing the sample.Plant the assembly together using a single tilt specimen holder at room temperature to form a hermetically sealed enclosure. Alternatively, use auto-loader grid clips and 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.The specimen is now ready to be inserted into the TEM. Examine samples placed in auto-loaders under cryo conditions or at room temperature. For liquid-EM imaging, load the specimen holder into the TEM equipped with a field emission gun and operate at 200 kilovolts.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 minus 15 degrees to plus 15 degrees in the column. This procedure adjusts the stage in the Z direction to accommodate the sample thickness and helps to ensure an accurate magnification during image recording. Record images as long framed movies or individual images using the serial data collection software package, implementing automated imaging routines.Acquire images under low-dose conditions at magnifications ranging from 28, 000X to 92, 000X and 40 frames per second. Adjust exposure times to minimize beam damage to the specimen and use a defocus range of minus one to four micrometers at the specified magnification. If a thick solution is encountered, use higher defocus values or select a different region of interest.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. Analyze movies for SARS-CoV-2 particles using the RELION-3.0.8 program or any other image processing software. Perform motion correction using MotionCor2 program.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. Calculate the initial reconstructions using C1 symmetry with the program's 3D initial model routine and the ab initio model options in the data processing software package.Then perform refinement protocols in the data processing software. Examine the results using a molecular structure analysis software package while assessing dynamic changes. A comparison of liquid-EM and cryo-EM structures in adeno-associated virus subtype three or AAV is shown here.The representative images show the structure of AAV in the solution and in ice. The rotational views of the AAV VP1 subunit extracted from the liquid and ice structures are shown here. These images represent the dynamic values in the liquid structures generated using the morph map function in the molecular structure analysis software.The average structures from multiple virus assemblies show conformational changes with an almost 5%diameter change measured using EM data. An image of SARS-CoV-2 subviral assemblies isolated from serum fractions from COVID-19 patients is shown here. These white bubbles indicate the presence of liquid in the sample.An EM reconstruction of these subviral assemblies is shown here with colored radial densities at five nanometer slices through the map. The representative image describes the analysis of rotavirus double-layered particles prepared in vitreous ice using the microchip sandwich technique. The use of detergents, glycerol, polyethylene, glycols, and high levels of sugars should be minimized or avoided for liquid-EM imaging.These reagents may introduce artifacts, create excessive bubbling, hydrolysis products, and free radicals due to beam damage. The use of these protocols will allow scientists to be able to study dynamic processes at atomic detail. And this spans across multiple fields of science including medicine, life sciences, and materials research.The protocols presented here describe how state-of-the-art tools can provide an exciting means to visualize biological macromolecules through new eyes. The liquid electron microscopy field may elevate how we study these novel viruses that pose a threat to human health, perhaps even contributing to our pandemic preparedness measures.

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

Research

JoVE Journal

Biochemistry

2.8K visualizações.  Pennsylvania State University. O interesse na microscopia eletrônica líquida disparou nos últimos anos, como podemos visualizar agora nos processos em tempo real em nanoescala. Um melhor conhecimento sobre essas estruturas flexíveis pode nos ajudar a desenvolver novos reagentes para combater patógenos emergentes, como o SARS-CoV-2. Ver proteínas em um ambiente fluido ajuda a imitar sistemas biológicos e nos dá a oportunidade de olhar para as proteínas de uma maneira mais dinâmica.E este experimento mostrar...