On-slide Preparation of Caenorhabditis elegans Towards Quantitative, High-Resolution LA-ICP-TOF Mass Spectrometry Imaging

Abstract Metal homeostasis is a complex process wherein essential metals serving structural, catalytic and regulatory roles are acquired, trafficked, and exported once they are present in excess. Understanding changes in metal content and localization in heterogenous tissue types is critical to understanding fundamental physiology as well as a growing number of disease states. Laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) imaging is a powerful technique for untargeted quantitation and mapping of metals in biological systems. While the nematode Caenorhabditis elegans ( C. elegans ) is a well-established model organism for fundamental biological research and metal-based diseases, there have been few reports of mass spectrometry-based imaging of C. elegans , mostly due to challenges preparing samples that maintain the native distribution of the elements. In this study, we developed an embedding, quantitation and imaging workflow that preserves C. elegans using 3D-printed uniform layer media application tools (ULMATs). Multiple embedding media were evaluated, and petrolatum, commercially known as Vaseline™, stood out for its performance in preserving C. elegans for imaging applications. Worms were subjected to microscopy and LA-ICP-TOF-MS imaging where we achieved a 2-μm spatial resolution by over-sampling laser shots during ablation. Quantitative elemental maps were obtained using a series of gelatin standards that were sectioned at a 40-μm thickness to closely mimic the average tissue ablation depth of a Day 1 gravid adult C. elegans . Our results establish a new workflow for comprehensive elemental profiling of C. elegans using LA-ICP-TOF-MS, which holds high potential for future spatial metal biology research with C. elegans . TOC