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

Toshihiko Ogura,Laurent Kreplak

doi:10.1371/journal.pone.0092780

Toshihiko Ogura, Laurent Kreplak

Open Access

https://doi.org/10.1371/journal.pone.0092780

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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.

Highlights

Scanning electron microscopy (SEM) is an important technique for producing high-resolution images of biological samples [1,2,3]
A measurement terminal under the sample holder detects the transmission of an electric frequency signal to the biological specimens in water
The irradiated electrons are scattered and absorbed in the W–Nicoated silicon nitride (SiN) film on the biological specimens; our method is capable of undamaged observation

Summary

Introduction

Scanning electron microscopy (SEM) is an important technique for producing high-resolution images of biological samples [1,2,3]. To allow observation under high vacuum conditions and to avoid electrical radiation damage by SEM, the biological specimens must be prepared using glutaraldehyde fixation, negative staining, a cryo technique, and/or a metal coating [4,5,6] These preparations have positive effects in terms of contrast enhancement and allowing the biological specimens to remain uncharged. Atmospheric and/or wet biological specimens have been observed using atmospheric holders [7,8,9,10] Using these methods, the specimens undergo heavy radiation damage caused by the electron beam (EB) [11,12,13], while unstained samples give a very poor contrast [9,10]. This system is effective to analyse the biological molecules in water layer

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Results

Conclusion