Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Podsumowanie

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7µm. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Streszczenie

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7µm. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

Protokół

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.

Dyskusje

In this presentation, we focused on the 3D-visualization of the internal anatomy of a chelicerate micro-arthropod. The synchrotron X-ray measurements allow a pixel-resolution of down to 0.3µm, depending on the size of the sample. Here, we have shown data with 0.7µm pixel-resolution. Generally, synchrotron X-ray tomography can be useful for analysing small biological materials (or tissues) with low X-ray attenuation. The pixel resolution almost reaches that of conventional light microscopy. The technique can b...