Since the demonstration of the visibility of single heavy atoms (CREWE et al., 1971) and unstained biological molecules (CREWE and WALL, 1970) using the scanning transmission electron microscope (STEM), biologists have been excited by the potential of the STEM for biological structure determination. Although heavy atom selective staining will probably become the most important future application of the STEM, several other applications are already well established and will be essential to development of selective staining. These applications derive from the incoherence (not to be confused with the common usage of this term) of the signal measured by the STEM annular detector, particularly if large angle scattering (40–200 mRadian) is recorded (FERTIG and ROSE, 1977). Coherence length is a measure of the distance between two scattering centers at which it is possible for the presence of the second center to influence scattering detected from the first. FERTIG and ROSE calculate that this coherence length for the conditions normally used in the STEM (.015 radian illumination half angle,.04–.20 radian annular detector acceptance, see Fig. 12.1) in the plane of the specimen is ~0.9 A and in the direction of the beam, ~30 A. Thus, it is unlikely that more than two atoms could give rise to coherent scattering, except in a crystalline specimen. This means that the STEM large angle annular detector signal is proportional to the number of atoms in the beam at any instant, the proportionality constant being the atomic scattering cross-section, σA(A2). It can be shown that if the specimen is scanned with a focused or defocused beam to give a uniform dose D(el/A2), the number of scattered electrons from a given atom will be D·σ, independent of the focal setting. This linearity between STEM large angle detector signal and specimen mass thickness makes quantitative interpretation of STEM images straightforward, even at high resolution. The major limitation to interpretability then becomes radiation damage.
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