The capacity to explore soft tissue structures in detail is important in understanding
animal physiology and how this determines features such as movement, behaviour and the impact
of trauma on regular function. Here we use advances in micro-computed tomography (micro-CT)
technology to explore the brain of an important insect pollinator and model organism, the
bumblebee (Bombus terrestris). Here we present a method for accurate imaging and exploration
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of insect brains that keeps brain tissue free from trauma and in its natural stereo-geometry, and
showcase our 3D reconstructions and analyses of 19 individual brains at high resolution.
Development of this protocol allows relatively rapid and cost effective brain reconstructions,
making it an accessible methodology to the wider scientific community. The protocol describes the
necessary steps for sample preparation, tissue staining, micro-CT scanning and 3D reconstruction,
followed by a method for image analysis using the freeware SPIERS. These image analysis methods
describe how to virtually extract key composite structures from the insect brain, and we
demonstrate the application and precision of this method by calculating structural volumes and
investigating the allometric relationships between bumblebee brain structures.