Understanding the morphology of anatomical structures is a cornerstone of biology. For small animals, classical methods such as histology have provided a wealth of data, but such techniques can be problematic due to destruction of the sample. More importantly, fixation and physical slicing can cause deformation of anatomy, a critical limitation when precise three-dimensional data are required. Modern techniques such as confocal microscopy, MRI, and tabletop x-ray
microCT provide effective non-invasive methods, but each of these tools each has limitations including sample size constraints, resolution limits, and difficulty visualizing soft tissue. Our research group at the Advanced Photon Source (Argonne National Laboratory) studies physiological processes in insects, focusing on the dynamics of breathing and feeding. To determine the size, shape, and relative location of internal anatomy in insects, we use synchrotron microtomography at the beamline 2-BM to image structures including tracheal tubes, muscles, and gut. Because obtaining naturalistic, undeformed anatomical information is a key component of our studies, we have developed methods to image fresh and non-fixed whole animals and tissues. Although motion artifacts remain a problem, we have successfully imaged multiple species including beetles, ants, fruit flies, and butterflies. Here we discuss advances in biological imaging and highlight key findings in insect morphology.