We are able to visualize the native structure of bacterial and archaeal cells thanks to a breakthrough in TEM sample preparation. Instead of getting rid of the water, why not just freeze it, since ice sublimates very slowly in a vacuum? The problem is that water expands as it freezes into crystalline ice, damaging the cell in the process. But in the 1980s scientists discovered that freezing a sample quickly enough (done by rapidly plunging a small volume into a very efficient cryogen like liquid ethane) creates a very different kind of ice. The water molecules are immobilized so abruptly that they do not have a chance to find binding partners to form a crystal. The result, called “vitreous” ice for its glass-like properties, preserves cells in their native, fully-hydrated state. The frozen sample can then be inserted directly into the vacuum of the TEM without needing additional treatment or staining. This technique is called cryogenic electron microscopy, or cryo-EM.
Here you see a projection image of a Caulobacter crescentus cell imaged by cryo-EM. Let us quickly go through how it was prepared. First a drop of culture was placed onto an EM grid. Instead of the glass slides used to support samples in a light microscope, EM sample supports are small circular grids of metal, ~3 mm across, overlaid by a thin mesh of carbon with 2 μm-wide holes. Excess liquid was then blotted away with paper, leaving a thin film of sample across the grid. The grid was then plunged into a cryogen, and the frozen sample was imaged in a special TEM that kept the sample cold (near the temperature of liquid nitrogen, approximately -190ºC) so that the vitreous ice did not warm and transition to a more damaging (and opaque) crystalline state. In this image, you can see the edge of one of the holes in the carbon mesh of the grid; note the slightly increased clarity in the hole where there is nothing but culture media compared to the region covered by carbon. Whenever possible, we choose to image cells lying at least partially in holes. You will also notice many small dark circles – these are gold beads that were added to the sample; you will see why on the next page.
With cryo-EM, we can start to see the true structure of cells. Compared to the images you have already seen, note the added level of detail visible here, including the cell’s multi-layered boundary, and the braided texture of its flagellum.
