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

Summary

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.

Abstract

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.

Protocol

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 ....

Discussion

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.......