1.6 Cryogenic Electron Tomography Atlas of Bacterial and Archaeal Cell Structure Home

Missing wedge

Think again about how a tilt-series is made: by taking images from different angles. If we could tilt the sample all the way to 90º, we would have information from every angle. But the sample gets effectively thicker as we tilt it, since the beam has to pass through more and more of the surrounding material. This effect, illustrated on the left, usually becomes prohibitive beyond 60º, so a typical tilt-series spans only ~2/3 of the possible angles, leaving a “missing wedge” of information corresponding to those high tilt angles, as you can see on the right. The missing wedge blurs densities in the direction of the imaging beam. In practice it means that if we look at a cross-section of a cell, we cannot trace thin features like membranes all the way around. You will see an example of this effect in Chapter 5.10.

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Cryogenic Electron Tomography

To truly understand a three-dimensional object, we need to be able to visualize it in three dimensions. To do that, we can use tomography (from the Greek for “writing slices”). The process may be familiar from medical Computed Tomography, or CT, scans. Simply, the object is imaged from different angles (in a CT scan, the camera moves around the patient; in our case we keep the imaging path constant and simply rotate the small sample). This produces a “tilt-series” of projection images that can be digitally processed into a 3D reconstruction of the object: a tomogram. To compute the reconstruction, we need to be able to precisely align the images, which is difficult because of the low contrast from cryo-EM samples (unstained and sensitive to electron dose). This is where the gold beads come in. As you saw on the previous page, they provide clear markers in the images to guide the alignment.

Here you see a tilt-series and resulting tomogram of a Caulobacter crescentus cell. In this and following movies, we view the tomogram as a series of slices scanning (or “writing”) from bottom to top. Note the further level of detail that this technique provides, separating the cell’s structures into their three-dimensional locations. We can also rotate the tomogram and slice along a different axis to view structures from different angles, although not all features are visible at all angles (⇩).

Since the early 2000s, cryo-ET has transformed our understanding of microbial cells, revealing their structural richness and diversity, as you will see in the chapters that follow.

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