High-Contrast Observation of Unstained Proteins and Viruses by Scanning Electron Microscopy

Toshihiko Ogura,Jianming Qiu

doi:10.1371/journal.pone.0046904

Abstract

Scanning electron microscopy (SEM) is an important tool for the nanometre-scale analysis of the various samples. Imaging of biological specimens can be difficult for two reasons: (1) Samples must often be left unstained to observe detail of the biological structures; however, lack of staining significantly decreases image contrast. (2) Samples are prone to serious radiation damage from electron beam. Herein we report a novel method for sample preparation involving placement on a new metal-coated insulator film. This method enables obtaining high-contrast images from unstained proteins and viruses by scanning electron microscopy with minimal electron radiation damage. These images are similar to those obtained by transmission electron microscopy. In addition, the method can be easily used to observe specimens of proteins, viruses and other organic samples by using SEM.

Highlights

To better understand various biological functions, observation of the nanometre structures of proteins and viruses is essential [1,2,3]
Scanning electron microscopy (SEM) System Overview Unstained proteins and viruses were deposited onto a metalcoated SiN film (Figure 1A)
The metal-coated SiN film, positioned with the SiN side up and the Au side down and with the sample mounted onto the downfacing Au side, is irradiated from above with a 4-kV electron beam (EB) from a field-emission SEM (FE-SEM)

Summary

Introduction

To better understand various biological functions, observation of the nanometre structures of proteins and viruses is essential [1,2,3]. Biological samples are prone to radiation damage and unstained samples give very poor contrast [6,7,8]. To address these problems, traditional sample preparation techniques such as glutaraldehyde fixation, negative staining and heavy metal coating have been developed [9,10,11] and used for several proteins and viruses [12]. The obtained images have very low contrast and high noise owing to the low radiation dose needed to minimize sample damage [6]. The goal of observing unstained proteins and viruses by electron microscopy under high contrast and with low radiation damage remains elusive

Methods

Results

Conclusion