Critical underfocus in low-magnification electron microscopy: rationale and practice.
Focus in transmission electron microscopy, especially at low-to-moderate powers, is usually achieved by an empirical, though largely hap-hazard, process of defocusing the objective below what is known to be perfect focus as defined by the absence of Fresnel diffraction fringes at the edges of holes. In this article we provide a precise, empirical method of focus, whose rationale resides in the less-than-perfect image transfer from the image space of the objective to the negative, i.e., the modulation transfer function. In practice, "perfect focus," as defined above, is established with a beam deflector ("wobbler"), to which underfocus is then applied routinely by reference to a table. Such "critical underfocus" values have to be calibrated, as described here, for each microscope and depend on all factors that influence contrast, that is, accelerating voltage, condenser defocus and coherence of illumination, focal length of the objective, aperture sizes, combination of intermediate and projection lenses, and the properties of the photographic emulsion.
Fahrenbach
W H
WH
Laboratory of Electron Microscopy, Oregon Regional Primate Research Center, Beaverton 97006.
Luft
J H
JH
D004655:Emulsions; D008854:Microscopy, Electron / Q000379:methods*; D008858:Microscopy, Phase-Contrast
D004655:Emulsions