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

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

Example of response to scanning in capture mode.

<p>The continuous images of one tick are shown. Time under vacuum conditions is shown on top of each picture. (A) An image of TV mode before capture. (B and C) The scanned images. (D) A captured image. Distortion of image is clearly observed at the pulvilus. (E and F) The images of TV mode after D.</p

Photograph of adult and nymph <i>H. flava</i>.

<p>(A and B) Photograph of a living adult female. Back and abdomen are shown. (C and D) A dead adult female. (E and F) A living nymph. (G and H) A dead nymph.</p

Motion of a live tick under SEM.

<p>Images of the TV mode were recorded. Time under vacuum conditions is shown on top of each picture. Leg movements are indicated by triangles.</p

Movement of four different ticks during image capture.

<p>(A–D) An example of second leg and third leg. Distortion is indicated by triangles. (E) Frequency of movement from multiple experiments is shown. Totally, 178 captures were performed using 20 nymph ticks. Each leg was captured more than 2 times. The capture numbers were; 43 times for 1st leg, 46times for 2^nd leg, 45 times for 3^rd leg and 44 times for 4th leg.</p

Survival of ticks after SEM observation.

<p>Nymph ticks were classified into three groups; exposed to vacuum with electron beam, exposed to vacuum and control with no treatment. Each group consists of eight ticks. % of survivors per tested group are shown.</p