DiceCT for Fishes: Recommendations for pairing iodine contrast agents with μCT to visualize soft tissues in fishes.

Computed tomography (CT) scanning and other high-throughput 3D visualization tools are transforming the ways we study morphology, ecology, and evolutionary biology research beyond generating vast digital repositories of anatomical data. Contrast-enhanced chemical staining methods, which render soft tissues radio-opaque when coupled with CT scanning, encompass several approaches that are growing in popularity and versatility. Of these, the various diceCT techniques that use an iodine-based solution like Lugol's have provided access to an array of morphological datasets spanning extant vertebrate lineages. This contribution outlines straightforward means for applying diceCT techniques to preserved museum specimens of cartilaginous and bony fishes, collectively representing half of vertebrate species diversity. We contrast the benefits of using either aqueous or ethylic Lugol's solutions and report few differences between these methods with respect to the time required to achieve optimal tissue contrast. We also explore differences in minimum stain duration required for different body sizes and shapes and provide recommendations for staining specimens individually or in small batches. As reported by earlier studies, we note a decrease in pH during staining with either aqueous or ethylic Lugol's. However, we could not replicate the drastic declines in pH reported elsewhere. We provide recommendations for researchers and collections staff on how to incorporate diceCT into existing curatorial practices, while offsetting risk to specimens. Finally, we outline how diceCT with Lugol's can aid ichthyologists of all kinds in visualizing anatomical structures of interest: from brains and gizzards to gas bladders and pharyngeal jaw muscles. This article is protected by copyright. All rights reserved.