Mass Spectrometry Imaging for Mapping the Spatial Distribution of Bioactive Compounds in Medicinal Fungi: Applications, Challenges, and Future Perspectives

Mass spectrometry imaging (MSI), featuring label-free detection, minimal sample pretreatment, and simultaneous multi-constituent analysis, overcomes the loss of spatial information inherent in conventional homogenization–extraction methods, enabling in situ visualization of bioactive constituents. This review systematically summarizes the recent advances and future perspectives of MSI in medicinal fungi, highlighting typical applications in Basidiomycota (e.g., Lentinula edodes, Ganoderma spp.) and Ascomycota (e.g., Ophiocordyceps sinensis, Cordyceps cicadae), which demonstrate the expansion of MSI from fundamental spatial metabolite localization to cultivar authentication and informing biosynthetic pathway investigations. We further analyze the critical challenges in medicinal fungal sample preparation—such as irregular morphological sizes and heterogeneous tissue structures—along with their resolution strategies. Subsequently, we outline the future directions of MSI, including ultra-high-resolution imaging, ion mobility integration, and spatial multi-omics fusion. Ultimately, MSI is propelling medicinal fungi research into a new era of “spatial metabolomics,” providing a crucial molecular spatial perspective for the precise exploitation of fungal resources, quality control, and the deep decoding of biosynthetic mechanisms.