In vivo X-ray 4D cine-tomography for tracking morphological dynamics in insects

The best method to understand the functional morphology of an arthropod joint is to observe its dynamics in 4D, that is in real-time and in 3D space. State-of-the-art optical microscopy techniques are confined to transparent or fluorescent objects, or are limited either by low penetration depth < 1 mm or poor time resolution. For insects and other optically opaque living organisms X-ray imaging methods are highly appropriate due to the penetration ability of the radiation.

Synchrotron radiation facilities provide brilliant radiation for volume imaging methods such as synchrotron-based X-ray computed microtomography (SR-µCT) that has already proven to be a powerful tool for investigating insect morphology in 3D. Real-time in vivo imaging of insects with high temporal resolution was realized so far by recording time sequences of 2D X-ray projection radiographs, obtaining time information about dynamics but losing any information about the third spatial dimension.

Aiming to investigate 3D morphological dynamics with feature sizes in the micron range and with temporal resolution down to a few tens of milliseconds, we designed a system for in vivo X-ray 4D cine-tomography. This technique comprises ultra-fast SR-µCT and 3D reconstruction, automated data processing and motion analysis procedures.

We demonstrate the potential of X-ray 4D cine-tomography by investigating the dynamics of screw-and-nut type hip joints in living granary weevils (Sitophilus granarius).