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

Abstract

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.

Keywords: brain; central nervous system; microCT; spiders; staining solution RRID:NCBITaxon_74971.

FIGURE 1

Specimen preparation for microCT scans including spider dissection, fixation, ethanol dehydration series, staining, ethanol washes, drying process, and scan medium (created with Biorender.com); (a) mounted specimen in liquid medium inside the MicroCT. CPD, critical point drying; PTA, phosphotungstic acid.

FIGURE 2

Small spiders: microCT scans of total prosoma sagittal view, with a zoomed‐in CNS and prosoma dorsal view: (A) Myrmarachne smaragdina; (B) Helpis sp.; (C) Xysticus bimaculatus. Scale bars: 1 mm. CPD, critical point drying; PTA, phosphotungstic acid.

FIGURE 3

Medium spiders: microCT scans of Hortophora biapicata, showing the effects of scan duration on scan quality. (A) Scan focused on whole prosoma, (B) Scan focused on whole prosoma with zoomed‐in CNS and (C) Scan focused on the brain. A 0.5 mm aluminium filter has been applied to all imaging. Views, from left to right, are sagittal view of the CNS; dorsal view at the arcuate body plane; dorsal view at the esophagus plane; and dorsal view at the VNC plane. Scale bar: 1 mm. PTA, phosphotungstic acid.

FIGURE 4

Large spiders: microCT scans of total prosoma sagittal sections, with zoomed‐in CNS and prosoma dorsal view: (A) Isopeda villosa, ethanol‐PTA; (B) Delena cancerides treated with Bouin‐iodine‐CPD. Scale bar: 1 mm. CPD, critical point drying; PTA, phosphotungstic acid.

FIGURE 5

Large spiders: comparison among spiders with dissection of the carapace or sternum. (A) Delena cancerides treated with Bouin‐iodine‐CPD with the sternum removed, (B) D. cancerides treated with ethanol‐PTA with the carapace removed, and (C) Isopeda villosa treated with ethanol‐PTA with the sternum removed. Scale bar: 1 mm. CPD, critical point drying; PTA, phosphotungstic acid.

FIGURE 6

Comparison of scan quality for short (left side) scans and long scans (right side) of small spiders Xysticus bimaculatus 10‐min scan (A) and 61‐min scan in aluminium tube (B), medium spiders Hortophora biapicata 10‐min scan (C) and 131‐min scan in 0.5 mm aluminium filter (D) and large spiders E: Isopeda villosa 10‐min scan (E) and 61‐min scan in 0.5 mm aluminium filter (F). All specimens were stained with ethanol‐PTA. Scale bar: 0.5 mm.