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Direct Observation of Unstained Biological Specimens in Water by the Frequency Transmission Electric-Field Method Using SEM

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  • Toshihiko Ogura

Abstract

Scanning electron microscopy (SEM) is a powerful tool for the direct visualization of biological specimens at nanometre-scale resolution. However, images of unstained specimens in water using an atmospheric holder exhibit very poor contrast and heavy radiation damage. Here, we present a new form of microscopy, the frequency transmission electric-field (FTE) method using SEM, that offers low radiation damage and high-contrast observation of unstained biological samples in water. The wet biological specimens are enclosed in two silicon nitride (SiN) films. The metal-coated SiN film is irradiated using a focused modulation electron beam (EB) at a low-accelerating voltage. A measurement terminal under the sample holder detects the electric-field frequency signal, which contains structural information relating to the biological specimens. Our results in very little radiation damage to the sample, and the observation image is similar to the transmission image, depending on the sample volume. Our developed method can easily be utilized for the observation of various biological specimens in water.

Suggested Citation

  • Toshihiko Ogura, 2014. "Direct Observation of Unstained Biological Specimens in Water by the Frequency Transmission Electric-Field Method Using SEM," PLOS ONE, Public Library of Science, vol. 9(3), pages 1-6, March.
    • Handle: RePEc:plo:pone00:0092780
    DOI: 10.1371/journal.pone.0092780
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    References listed on IDEAS

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    1. Toshihiko Ogura, 2012. "High-Contrast Observation of Unstained Proteins and Viruses by Scanning Electron Microscopy," PLOS ONE, Public Library of Science, vol. 7(10), pages 1-7, October.
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    Cited by:

    1. Toshihiko Ogura, 2019. "Direct observation of unstained biological samples in water using newly developed impedance scanning electron microscopy," PLOS ONE, Public Library of Science, vol. 14(8), pages 1-17, August.
    2. Toshihiko Ogura, 2022. "Development of multi-frequency impedance scanning electron microscopy," PLOS ONE, Public Library of Science, vol. 17(1), pages 1-15, January.

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