Visualizing Heat-Treated Viral Capsids Using Transmission Electron Microscopy

Take tubes containing viral capsids, which are virus-like particles without genetic material.

Heat the tubes at a constant temperature for varying durations.

A shorter heating duration mildly stresses the capsid proteins, while prolonged heating disrupts the capsid and causes aggregation.

Rapidly cool the tubes to prevent further structural changes.

Transfer the liquid onto a paraffin film.

Place a transmission electron microscopy (TEM) grid onto the droplet. The capsids attach to the grid's surface.

Blot the grid with a filter paper to remove excess fluid.

Wash with a buffer to remove loosely attached capsids, then blot the grid again.

Dip in uranyl acetate, a heavy metal stain that binds to the capsid surface, making them appear darker under TEM.

Blot the grid and place it on a plate. Store the plate in a desiccator to remove moisture.

Image the grid using TEM to visualize heat-induced capsid damage.

Perform heat treatment as before except substitute PBS for 10 millimolar HEPES, pH 7.4 and do not further dilute capsids after treatment. Next, pipette the entire 15 microliter drop of treated capsid solution onto paraffin film. Grab the grid edge with tweezers allowing them to close on the edge to hold the grid and place grid carbon side down on top of the drop of treated solution.

Let the grid incubated for 10 minutes to allow the capsid to attach to the grid. Depending on the purity of the capsid preparation, add washes of the HEPES solution in the form of 10 to 15 microliter droplets placed in line after the treated capsid droplet. After 10 minutes, pick up the grid with the droplet.

Place the grid nearly perpendicular to a piece of filter paper to wick away the droplet. Then, use the grid to pick up the droplet of 10 to 15 microliter HEPES buffer and hold it for 30 seconds. After 30 seconds of wash time, wick away with another piece of filter paper.

Next, place a 15 microliter droplet of two percent uranyl acetate solution on the paraffin film in an empty area. Pick up the droplet of uranyl acetate with the grid and hold it for 45 seconds. Then, wick away the uranyl acetate being sure to remove most of the solution.

Place the grid carbon side up on a piece of filter paper being careful that the grid does not stick to the moisture on the tweezers. Then, place the filter paper with the grid on it in an open glass Petri dish. After repeating this process for all treatments, place the Petri dish halves with the grids in a desiccator overnight to dry prior to observing with TEM.