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

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.