This book (Practical Electron Microscopy and Database) is a reference for TEM and SEM students, operators, engineers, technicians, managers, and researchers.

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As discussed in page2779, the contrast of atom columns in HRTEM images can be switched between black, white and gray with defocusing function.

The intensity distribution of Fresnel fringes in TEM images varies with the defocus. There is a significant difference between slightly overfocused and underfocused images. Especially, the underfocused image shows an extremely high contrast up to I_max/I_min ~ 3. Here, is the maximum and the minimum intensities.

Figure 4214 shows the TEM images of 1-nm He (helium) bubbles in palladium tritides when the specimen is oriented far from any Bragg conditions of the matrix of the palladium tritides [1]. For negative defocus (underfocus), the bubbles appear as white dots surrounded by a dark fringe, while for positive defocus (overfocus), the dots are black with white fringe.

Figure 4214. The TEM images of 1-nm He (helium) bubbles in palladium tritides: (a) Underfocus and (b) Overfocus. The insets present theoretically simulated TEM contrasts of centered He bubbles in 6.5 nm thick TEM specimen. Adapted from [1]

Due to the positive spherical aberration in traditional TEMs, the only way to produce phase contrast from a thin object is to use an underfocus setting. The resulting aberration function from underfocus is negative and the phase change is positive. The positive phase contrast from a weak phase object is thus dark with respect to the background (mean intensity).

TEM images of thin biological, frozen-hydrated specimens must be recorded with a substantial degree of defocus in order to generate adequate contrast at low spatial frequencies.