Electron microscopy has often been used to visualize arrays of globular macromolecules within various cell preparations. For example, in the outer membranes of mitochondria, details were enhanced by use of a negative stain such as phosphotungstate (e.g., Parsons, 1967). A significant conceptual breakthrough occurred when crystallographic principles were adapted to the analysis of images of periodic objects, e.g., the pioneering work of DeRosier (1971) and DeRosier and Klug (1968) on T4 bacteriophage tail. (For a discussion of tubular globular protein crystals, see Vainshtein (1978).) In this work, an average image of the object was reinforced by spatially filtering the signal from the repeating motif. Such filtering could be carried out on an optical bench, using a physical mask to pass (through drilled or etched holes) the intense maxima in the Fourier transform formed at the back focal plane of the objective lens. When the continuous signal, due to the nonperiodic part of the array, was removed, a clearer image would be obtained (e.g., see Misell, 1978). Later this work was carried out digitally, using computer software that would allow calculation of forward and reverse Fourier transforms. Insertion of the mask function was applied when the reverse transform to the image was computed (e.g., Van Heel and Keegstra, 1981) or, alternatively, just the centers of the maxima were used to obtain the phase terms (e.g., Hovmöller, 1992). Correlation techniques were developed, not only for periodic objects, but also nonperiodic ones, so that small arrays could be aligned to one another to form an average by superposition (Frank, 1980; Saxton, 1980; Saxton and Baumeister, 1982).
KeywordsElectron Diffraction Pattern Phase Error Phase Extension Diffuse Signal Optical Bench
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