Micro‐CT visualization of the CNS: Performance of different contrast‐enhancing techniques for documenting the spider brain

Spider brain and central nervous system (CNS) have remained unexplored, due in part to the difficulty of observing these organs, usually only possible through histological preparations. Recently, internal anatomy studies have been supplemented by the inclusion of X‐ ray micro‐CT. Unmineralized tissue such as the body of invertebrates requires a staining process to enhance tissue X‐ray absorption and improve contrast during observation. Many current protocols are based on iodine staining requiring critical point drying (CPD) of the sample for optimal contrast. This process induces shrinking of the soft tissue generating artifacts in the morphology, volume, and even position of internal structures. Phosphotungstic acid (PTA) is an alternative staining agent recently used in marine invertebrate and plant studies. Here, we used several specimens of the common spider Araneus diadematus to visualize the spider brain and compare both contrast‐enhancing ethanol‐based solutions. We assessed a gradient of staining times, observed and tested the repercussions of CPD, and examined the use of vacuum to accelerate PTA diffusion. We show that PTA provides the best contrast on micro‐CT scans in ethanol eliminating the need for CPD, and offering more realistic in situ visualizations of the internal organs. In combination with different scanning settings, PTA allowed observation of internal organs like the CNS, digestive system, muscles, and finer structures like the retina, visual nerves, and optic neuropiles. This fast and less invasive method could facilitate the proper documentation of the internal anatomy in the context of evolutionary, developmental and functional studies.