Animals used in this study

Specimens of the parthenogenetic oribatid mite Archegozetes longisetosus (Acari, Oribatida) were taken from our laboratory culture. The culture grows on a plaster of Paris/charcoal mix (9:1) in plastic jars, in constant dark at 20-23°C with approximately 90% of air humidity.

Sample preparation

1. Specimens were taken from the culture, cleaned with a fine brush and placed in a 6:3:1 mixture of 80% ethanol, 35% formaldehyde and 100% acetic acid for 24 hours.
2. Afterwards, specimens were dehydrated in a graded ethanol series of 70%, 75%, 80%, 85%, 90%, 95%, and 100% with 3 changes at each concentration, and 10 min between the steps.
3. Finally, samples were placed in fresh 100% ethanol overnight and critical point dried in CO₂ (CPD 020, Balzers). Dried specimens were attached to the tip of plastic pins (1.2 cm long; 3.0 mm in diameter).

Synchrotron X-ray tomography

X-ray tomography was performed at beamline ID19 (ESRF, Grenoble, France, experiment SC-2127).

1. The samples were mounted in the sample-holder and adjusted to a central position in the beam.
2. Samples were measured with an energy of 20.5 keV. The radiographs were recorded with a cooled CCD (ESRF FReLoN camera) with a 14-bit dynamic range, 2048×2048 pixels and an effective pixel size of 0.7 μm. 1500 projections were recorded over the 180° sample rotation with an exposure time of 0.35 s for each projection. The detector-to-sample distance was 20 mm.

Using a certain distance between sample and detector enables a differential imaging of materials with low X-ray attenuation coefficients (Cloetens, et al. 1996), which would produce insufficient contrast in absorbtion imaging (where the sample is located directly in front of the detector). Most biological matters are phase objects, composed of materials with low absorbtion and/or only small differences in atomic number (Betz, et al. 2007). However, phase enhanced tomography requires a high spatial coherence of a homogeneous X-ray beam. Therefore, synchrotron radiation is better suited than desktop-scanners for these kind of measurements.

Data analysis

1. The resulting 2D radiographs were transformed to 3D voxel data (8-bit grey-values) with a filtered back-projection algorithm (Cloetens, et al., 1997)
2. The voxel data were analysed with the software VGStudio Max 1.2.1. (Volume Graphics, Heidelberg, Germany).
3. Grey-values from the background were removed from the histogram for 3D-visualization.
4. Pre-defined camera-paths were used to generate rotational animations and animated clipping planes.
5. A user-defined camera path was generated to follow the digestive system of A. longisetosus.