Although the first commercial scanning electron microscope (SEM) was introduced in 1965, the limited resolution and the lack of preparation techniques initially confined biological observations to relatively low magnification images showing anatomical surface features of samples that withstood the artifacts associated with air drying. As the design of instrumentation improved and the techniques for specimen preparation developed, the SEM allowed biologists to gain additional insights not only on the external features of samples but on the internal structure of tissues as well. By 1985, the resolution of the conventional SEM had reached 3 - 5 nm; however most biological samples still required a conductive coating of 20 - 30 nm that prevented investigators from approaching the level of information that was available with various TEM techniques. Recently, a new SEM design combined a condenser-objective lens system with a field emission electron source.
A scanning electron microscope using a field emission electron source and a single electromagnetic lens can produce a resolution of less than 180Å using an accelerating voltage of only 900v. High resolution, low voltage (0.1-2kV) scanning microscopy offers a number of advantages over the use of higher accelerating voltages. Specimen damage may be reduced because the power (P≃IV) which must be absorbed by the specimen for operation at a given probe current (I) is decreased in proportion to the reduction in accelerating voltage (V).
Field emission scanning electron microscope (FESEM) features extremely high resolution images, and offers many valuable information. But, for a specimen which gives low contrast images, lateral stripes appear in images. These stripes are resulted from signal fluctuations caused by probe current noises. In order to obtain good images without stripes, the fluctuations should be less than 1%, especially for low contrast images. For this purpose, the authors realized a noise compensator, and applied this to the FESEM.Fig. 1 shows an outline of FESEM equipped with a noise compensator. Two apertures are provided gust under the field emission gun.
Even though many early workers had suggested the use of lower voltages to increase topographic contrast and to reduce specimen charging and beam damage, we did not usually operate in the conventional scanning electron microscope at low voltage because of the poor resolution, especially of bioligical specimens. However, the development of the “in-lens” field emission scanning electron microscope (FESEM) has led to marked inprovement in resolution, especially in the range of 1-5 kV, within the past year. The probe size has been cumulated to be 0.7nm in diameter at 30kV and about 3nm at 1kV. We have been trying to develop techniques to use this in-lens FESEM at low voltage (LVSEM) for direct observation of totally uncoated biological specimens and have developed the LVSEM method for the biological field.
Minimum feature sizes in experimental integrated circuits are approaching 0.5 μm and below. During the fabrication process it is usually necessary to be able to non-destructively measure the critical dimensions in resist and after the various process steps. This can be accomplished using the low voltage SEM. Submicron linewidth measurement is typically done by manually measuring the SEM micrographs. Since it is desirable to make as many measurements as possible in the shortest period of time, it is important that this technique be automated.Linewidth measurement using the scanning electron microscope is not well understood. The basic intent is to measure the size of a structure from the secondary electron signal generated by that structure. Thus, it is important to understand how the actual dimension of the line being measured relates to the secondary electron signal. Since different features generate different signals, the same method of relating linewidth to signal cannot be used. For example, the peak to peak method may be used to accurately measure the linewidth of an isolated resist line; but, a threshold technique may be required for an isolated space in resist.
“Wien–type” Filtered Secondary Electron Imaging of Immunocytochemically Labeled Methacrylate Sections of Tissues in a Field Emission Scanning Electron Microscope (FESEM)