Transmission Electron Microscopy
In Transmission Electron Microscopy (TEM), we detect electrons that have interacted with atoms in the sample as they passed through it, producing a “projection” image of the 3D object onto a 2D plane, similar to a medical X-ray image. This shows details throughout the cell, not just on the surface.
Electron microscopy, whether SEM or TEM, relies on the interactions of electrons with biological material to create an image. These interactions, however, also present some problems. First, they damage the sample, so exposure has to be limited, which in turn limits the contrast of images, or how much signal we see relative to noise. Second, electrons interact not just with the sample, but also with anything else in their path, so imaging must be conducted in a vacuum. This is problematic for biological material, which is mostly water that instantly boils away in a vacuum. To circumvent these problems, we can dehydrate samples to remove the water (changing the structure in the process) and coat them with metal to increase electron dose tolerance and contrast. The Shewanella oneidensis cells you saw on the last page were coated with platinum before imaging. In TEM, samples are often coated with a “negative stain” such as uranyl acetate; the electron-dense (dark in an image) metal pools around the sample, leaving the interior lighter and thereby creating a negative image. This S. oneidensis cell was negative-stained this way before imaging. The resulting projection image again shows the shape of the cell and its flagellum, but not many internal details.
There are more elaborate (and structure-altering) sample preparation methods for TEM, involving “fixing” the sample by chemical crosslinking or freezing under high pressure, dehydrating it, embedding it in resin, staining it, and slicing it into thin sections. With these methods, electron microscopists were able to discover many details of eukaryotic cells such as their internal organelles. Bacteria and archaea are much smaller, though, and lack many robust prominent structures like organelles. As a result, until the twenty-first century, we thought bacteria and archaea were structurally unexciting, little more than water balloons filled with small molecules. How wrong we were.