Comparing microCT Staining and Scanning Methodology for Brain Studies in Various Sizes of Spiders

Recent advances in microCT are facilitating the investigation of microstructures in spiders and insects leading to an increased number of studies investigating their neuroanatomy. Although microCT is a powerful tool, its effectiveness depends on appropriate tissue preparation and scan settings, particularly for soft, non-sclerotized tissues, such as muscles, organs, and neural tissues. As the application of microCT in spiders is only in its infancy, published protocols are often difficult to implement due to substantial size variation of the specimens. The present study was initiated to determine how to account for this variation. Our work builds on previous methods using microCT to image spider brains, with the aim to consolidate current knowledge and reduce time spent troubleshooting appropriate methodology, thereby facilitating future studies of spiders and their central nervous systems (CNS). We tested three different preparation and imaging techniques based on published protocols with minor modifications using 216 spiders with prosoma lengths ranging from 1.25 mm (small spiders) to 13.33 mm (large spiders). We compared the efficacy of the various specimen preparations, staining methods, and scan settings by categorizing the quality of dorsal and lateral microCT scans. We observed that only the phosphotungstic acid (PTA) staining agent resulted in complete staining of the prosoma and the CNS, allowing the CNS structures to be distinguished for small, medium, and large spiders. The use of image averaging, increased number of projections, image exposure timing, and detector binning did not greatly affect image quality for small and larger spiders but reduced noise. These settings did help improve image quality for medium spiders in conjunction with higher resolutions and an aluminum filter. We discussed the suitability of methods concerning spider size, effort, chemical risk, and image quality.