High-Resolution, Low-Voltage SEM of Cell Wall Regeneration of Yeast Shizosaccharomyces Pombe Protoplasts

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.

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Limits of SEM Resolution

Microscopy Today ◽

10.1017/s1551929500068929 ◽

1996 ◽

Vol 4 (5) ◽

pp. 10-11

Author(s):

David C. Joy

Keyword(s):

Electron Microscope ◽

Scanning Electron Microscope ◽

Secondary Electron ◽

Mechanical Stability ◽

Electron Sources ◽

The Past ◽

Probe Size ◽

Order Of Magnitude ◽

Imaging Mode ◽

Scanning Electron

Over the past decade the achievable resolution of the scanning electron microscope (SEM) in secondary electron (SE) imaging mode has improved by about one order of magnitude. In fact, instruments capable of demonstrating a resolution of one nanometer and exhibiting a probe size of less than 0.6 nm are not available. Continued improvements in electronoptics, electron sources, and in electronic and mechanical stability promise even smaller probe sizes that still contain adequate current for imaging, it is therefore relevant to consider what resolution might ultimately be achievable with an SEM in SE mode.

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Linewidth measurement using the low voltage SEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100144681 ◽

1986 ◽

Vol 44 ◽

pp. 652-653

Author(s):

M.G. Rosenfield

Keyword(s):

Electron Microscope ◽

Scanning Electron Microscope ◽

Integrated Circuits ◽

Secondary Electron ◽

Low Voltage ◽

Fabrication Process ◽

Critical Dimensions ◽

Linewidth Measurement ◽

Threshold Technique ◽

Scanning Electron

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.

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Comparison of freeze-fractured yeast replicas using conventional TEM and low-voltage field-emission SEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100152343 ◽

1989 ◽

Vol 47 ◽

pp. 74-75

Author(s):

William P. Wergin ◽

Eric F. Erbe ◽

Terrence W. Reilly

Keyword(s):

Electron Microscope ◽

Scanning Electron Microscope ◽

Field Emission ◽

Low Voltage ◽

Objective Lens ◽

Specimen Preparation ◽

Lens System ◽

Air Drying ◽

Preparation Techniques ◽

Scanning Electron

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.

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