Abstract
Abstract Recent improvements in digital image acquisition, and automated TEM, have lowered the electron dose requirement for collecting a tomographic tilt series to the point where cryo-electron tomography is now feasible. Theoretical calculations indicate that resolution values of 2-5 nm can be obtained for fully hydrated specimens 100-500 nm thick. This resolution is sufficient for identifying individual macromolecules, and macromolecular assemblies, within-organelles and small cells. However, initial applications to cells and organelles fell short of theoretical resolution limits, indicating further development is necessary before cryo-electron tomography reaches its full potential for characterizing macromolecules in situ. Axonemes are ideal test specimens for cryo-tomography because of their appropriate size (diameter of 250 nm), ease of isolation, and unique construction from regular arrays of well-characterized components. The latter include microtubules, radial spokes, central sheath components, outer dynein arm, inner dynein arms, and the microtubule subunit lattice. These components are convenient benchmarks for evaluating the actual detection limit of tomographic reconstructions within a resolution range of 3-30 nm. Furthermore, axoneme components are sufficiently well ordered that computed diffraction patterns contain layer-lines that are useful for judging the quality of individual 2D images.
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