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

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.