X-Ray Microscopy of the Larval Crustacean Brain

Micro-computed X-ray tomography (μCT) coupled with visualization techniques such as three-dimensional reconstruction of internal morphological structures has opened up new pathways for analyzing the anatomy of nervous systems in intact specimens. The possibility for combining μCT with other techniques is one of the major advantages of μCT scanning, and the technical development of higher resolutions in lab-based μCT-scanners allows for investigating the anatomy of specimens in the sub-milimeter range. The European shore crab Carcinus maenas features a larval development over four zoeal and one megalopal stage with body lengths ranging from 500 μm to 2000 μm. The developing nervous system in the larvae of C. maenas is organized into a central brain which is connected via esophageal connectives with a ventral nerve chord and segmental ganglia. Since soft tissues such as the nervous tissues feature low contrasts compared to other tissues such as muscles or cuticularized body parts, the interpretation in μCT scans is challenging and needs some practice. The protocol described here is also applicable for larger specimens of a variety of species and spans over 2-3 days resulting in an image stack ready for postprocessing and visualization.