Observation of Live Ticks (Haemaphysalis flava) by Scanning Electron Microscopy under High Vacuum Pressure

Yasuhito Ishigaki,Yuka Nakamura,Yosaburo Oikawa,Hideaki Nakagawa,Susumu Kuwabata,Yasuhiro Yano,Tsutomu Takegami,Naohisa Tomosugi,Luciano A Moreira

doi:10.1371/journal.pone.0032676

Abstract

Scanning electron microscopes (SEM), which image sample surfaces by scanning with an electron beam, are widely used for steric observations of resting samples in basic and applied biology. Various conventional methods exist for SEM sample preparation. However, conventional SEM is not a good tool to observe living organisms because of the associated exposure to high vacuum pressure and electron beam radiation. Here we attempted SEM observations of live ticks. During 1.5×10−3 Pa vacuum pressure and electron beam irradiation with accelerated voltages (2–5 kV), many ticks remained alive and moved their legs. After 30-min observation, we removed the ticks from the SEM stage; they could walk actively under atmospheric pressure. When we tested 20 ticks (8 female adults and 12 nymphs), they survived for two days after SEM observation. These results indicate the resistance of ticks against SEM observation. Our second survival test showed that the electron beam, not vacuum conditions, results in tick death. Moreover, we describe the reaction of their legs to electron beam exposure. These findings open the new possibility of SEM observation of living organisms and showed the resistance of living ticks to vacuum condition in SEM. These data also indicate, for the first time, the usefulness of tick as a model system for biology under extreme condition.

Highlights

Scanning electron microscopes (SEM), which image sample surfaces by scanning them with an electron beam, are widely used for steric observations of resting samples in various fields
The ticks survived the SEM observation under vacuum condition, and we succeeded in recording their movement as a video
The ticks were resistant to vacuum under our experimental condition (1023 Pa for 30 minutes), but seemed to be sensitive to electron beam irradiation used to obtain the images (Fig. 4)

Summary

Introduction

Scanning electron microscopes (SEM), which image sample surfaces by scanning them with an electron beam, are widely used for steric observations of resting samples in various fields. SEM has been widely used since the 1970s to observe ticks and mites in acarology because it enables the observation of surfacefine structure. Recent applications of low-temperature SEM analysis enabled observation of new morphological attributes and proposed a new model of leg locomotion [8,9]. These observations did not involve living organisms or real movements. We could cross-breed mites and ticks and determine the species’ characteristics more precisely if they survive after SEM observation. Until now, achieving this has been believed to be extremely difficult

Methods

Results

Conclusion