Sample preparation optimization of insects and zebrafish for whole-body mass spectrometry imaging

Appropriate sample preparation is one of the most critical steps in mass spectrometry imaging (MSI), which is closely associated with reproducible and reliable images. Despite that model insects and organisms have been widely used in various research fields, including toxicology, drug discovery, disease models, and neurobiology, a systematic investigation on sample preparation optimization for MSI analysis has been relatively rare. Unlike mammalian tissues with satisfactory homogeneity, freezing sectioning of the whole body of insects is still challenging because some insect tissues are hard on the outside and soft on the inside, especially for some small and fragile insects. Herein, we systematically investigated the sample preparation conditions of various insects and model organisms, including honeybees ( Apis cerana ), oriental fruit flies ( Bactrocera dorsalis ) , zebrafish ( Danio rerio ), fall armyworms ( Spodoptera frugiperda ), and diamondback moths ( Plutella xylostella ), for MSI. Three cutting temperatures, four embedding agents, and seven thicknesses were comprehensively investigated to achieve optimal sample preparation protocols for MSI analysis. The results presented herein indicated that the optimal cutting temperature and embedding agent were −20 °C and gelatin, respectively, providing better tissue integrity and less mass spectral interference. However, the optimal thickness for different organisms can vary with each individual. Using this optimized protocol, we exploited the potential of MSI for visualizing the tissue-specific distribution of endogenous lipids in four insects and zebrafish. Taken together, this work provides guidelines for the optimized sample preparation of insects and model organisms, facilitating the expansion of the potential of MSI in the life sciences and environmental sciences. Graphical Abstract