Micro-computed tomography of termite gut anatomy: effects of staining regime on resolvability

Examining anatomical features of exceptionally small animals presents significant challenges for researchers. Micro-Computed Tomographic (µ-CT) scanning coupled with target tissue staining has enabled many previously unseen structures of small insects to be mapped and measured in detail. Here we investigated the effectiveness of three staining regimes: Lugol's iodine solution (LS), LS plus 1% phosphotungstic acid (PTA), and LS plus 2% PTA, in enhancing the resolvability of µ-CT scans of the gut organs of the termite Nasutitermes exitiosus, which has protist-independent digestion. We compared pixel intensity and probabilistic tissue contrast (PTC) across the three staining regimes to determine their ability to enhance image contrasts and improve the resolvability of the internal structures. We measured and compared volume and surface area of the termite foregut, midgut, hindgut, salivary glands and cephalic ganglion (brain). Our results showed LS plus PTA significantly improved image resolution for these termite organs, with LS combined with 2% PTA being the most effective staining regime for enhancing pixel intensity and PTC of most of the organs studied. LS plus 1% PTA resulted in an improved image quality but was generally less effective than LS plus 2% PTA. The LS alone regime gave the lowest resolvability, especially for less radio-dense structures like the salivary glands and midgut. Our results suggest that staining with LS plus 2% PTA generally offers significant image enhancement of µ-CT derived images of microscopic insect organs. This approach can be applied more broadly to significantly improve visualizations of the internal anatomy in other small arthropods and soft-bodied invertebrates, facilitating future research in comparative morphology, developmental biology, and functional anatomy.