Tilt dependence of the secondary electron emission at low excitation energy

Influence of the specimen's slope on the secondary electron emission has been experimentally studied. Strong deviations from the inverse cos law have been observed and corresponding phenomenological equation (taking into account this deviation) is suggested. The consequences of the dependence on the topography contrast of low- and high-voltage scanning electron microscopy (SEM) image, especially for three-dimensional (3-D) reconstruction, are considered.     

Radiation damage relative to transmission electron microscopy of biological specimens at low temperature: a review.

When biological specimens are irradiated by the electron beam in the electron microscope, the specimen structure is damaged as a result of molecular excitation, ionization, and subsequent chemical reactions. The radiation damage that occurs in the normal process of electron microscopy is known to present severe limitations for imaging high resolution detail in biological specimens. The question of radiation damage at low temperatures has therefore been investigated with the view in mind of reducing somewhat the rate at which damage occurs. The radiation damage protection found for small molecule (anhydrous) organic compounds is generally rather limited or even non-existent. However, large molecular, hydrated materials show as much as a 10-fold reduction at low temperature in the rate at which radiation damage occurs, relative to the damage rate at room temperature. In the case of hydrated specimens, therefore, low temperature electron microscopy offers an important advantage as part of the overall effort required in obtaining high resolution images of complex biological structures.     

Application of ion-beam etching techniques to the fine structure of biological specimens as examined with a field emission SEM at low voltage

The term “etching,” in electron microscopy, refers to the removal of specimen surface layers and includes chemical, electrolytic, and ion-beam methods. The ion-beam etching process is used to remove layers of a target material by bombarding it with ionized gas molecules. Recently, the method has been applied to the field of biological specimens; however, the practical procedures for such organic materials have not been developed. In the present study, we used an apparatus in which a beam of argon ions is collimated and focused by electrostatic lenses onto an appropriate target. We demonstrated the optimum conditions to observe biological specimens that were treated with osmium tetroxide and tannic acid. The specimens were examined uncoated at low accelerating voltage using a field emission scanning electron microscope. According to our experiments, when a biological specimen was observed under high-resolution conditions at over 50,000x magnification, the optimum condition of ion-beam etching consisted of an accelerating voltage of E = 1 keV and an ion-beam dose of It = 360 ～ 400 μA. min, depending on parts of the specimens. In order to decrease overetching, we had to choose factors such as E = 1 ～ 2 keV and It = 500 μA. min.