Splitting Hairs: Optimized Sample Preparation Strategy for Mass Spectrometry Imaging of Nematode Caenorhabditis elegans

Jacobson, R. E.; Smith, E. W.; Saitoh, Y.; Qiu, T.

doi:10.1101/2025.12.01.691628

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bioRxiv · 10.1101/2025.12.01.691628

Splitting Hairs: Optimized Sample Preparation Strategy for Mass Spectrometry Imaging of Nematode Caenorhabditis elegans

Jacobson, R. E.·Smith, E. W.·Saitoh, Y.·Qiu, T.

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Abstract

Caenorhabditis elegans is a powerful model organism for studying development, neurobiology, aging, and toxicology, yet most investigations rely on genetic and phenotypic measurements that do not directly capture their biochemical landscape. Mass spectrometry (MS)-based metabolomics has begun to address this gap by enabling detection of small-molecule metabolites and lipids. However, conventional liquid or gas chromatography-MS approaches require homogenization of whole worms, eliminating spatial information. Mass spectrometry imaging (MSI) offers an avenue for visualizing metabolite distributions in situ, but its application to C. elegans is hindered by the nematodes small size and thick cuticle which make it challenging for structure preservation during cryo-sectioning and ion image interpretation. We develop an integrated sample preparation workflow to overcome these challenges and enable high-resolution MSI of C. elegans adult hermaphrodites. To facilitate anatomical and ion images interpretation, we employed a dual-fluorescent reporter strain expressing GFP in intestinal cells and RFP in neuronal nuclei. We optimized embedding media compositions and freezing strategies to preserve tissue architecture and genetically encoded reporter fluorescence during cryo-sectioning. Additionally, we implemented a simple "sandwiching" method that reproducibly orients worms, allowing consistent longitudinal sectioning. Using this optimized workflow, we achieved matrix-assisted laser desorption/ionization (MALDI) MSI of 10-m spatial resolution and demonstrated tissue-specific distributions of lipids and metabolites in adult hermaphrodites. With structural preservation, consistent orientation, and fluorescence-guided spatial annotation, our approach provides a foundation for investigating metabolic heterogeneity in C. elegans and can be adapted to other microscale biological systems for MSI applications.

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Jacobson, R. E., Smith, E. W., Saitoh, Y., Qiu, T.. 2025-12-01. Splitting Hairs: Optimized Sample Preparation Strategy for Mass Spectrometry Imaging of Nematode Caenorhabditis elegans. https://doi.org/10.1101/2025.12.01.691628

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