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Since its inception in 2004, DESI\u2010MSI has evolved through innovations in ionization sources, sprayer design, and data processing, allowing high\u2010throughput visualization of spatial distributions for diverse molecules (e.g., lipids, metabolites, drugs) in tissues. This review systematically reviews the fundamental principles, instrumental configurations, and critical technical parameters of DESI\u2010MSI. Recent technological advancements, specifically the development of nanoscale DESI achieving spatial resolutions under 50\u2009\u03bcm and the implementation of additive\u2010enhanced solvents such as metal ions and reactive anions to enhance sensitivity and specificity, are critically evaluated. Furthermore, the multidisciplinary applications of DESI\u2010MSI are comprehensively examined: (1) in biomedicine, it facilitates cancer margin delineation (e.g., breast, prostate tumors) and neuropathological biomarker discovery; (2) in pharmaceutical research, it enables in\u00a0situ drug distribution analysis and tissue metabolism profiling; (3) in plant sciences, it supports spatial mapping of phytochemicals, pesticide residues, and host\u2013microbe interactions. Nevertheless, despite its advantages in real\u2010time analysis and nondestructive sampling, inherent challenges persist, including limited spatial resolution (~200\u2009\u03bcm) and complexities in interpreting complex datasets. Future work should emphasize the integration of multimodal imaging approaches, machine learning\u2013driven data processing pipelines, and clinical translation strategies for intraoperative diagnostics. Collectively, this study positions DESI\u2010MSI as a transformative spatial metabolomics tool with expanding potential in clinical diagnostics and high\u2010throughput drug screening.<\/jats:p>","DOI":"10.1002\/jms.70004","type":"journal-article","created":{"date-parts":[[2025,12,30]],"date-time":"2025-12-30T09:09:38Z","timestamp":1767085778000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Desorption Electrospray Ionization Mass Spectrometry Imaging: Principles, Advancements, and Multidisciplinary Applications"],"prefix":"10.1002","volume":"61","author":[{"given":"Yan","family":"Wang","sequence":"first","affiliation":[{"name":"School of Pharmaceutical Sciences &amp; Institute of Materia Medica, Shandong First Medical University &amp; Shandong Academy of Medical Sciences  Jinan China"},{"name":"School of Pharmacy Jining Medical University  Rizhao China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huannan","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Pharmacy Jining Medical University  Rizhao China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuncun","family":"Zhang","sequence":"additional","affiliation":[{"name":"School of Pharmacy Jining Medical University  Rizhao China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yidan","family":"Xi","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences &amp; Institute of Materia Medica, Shandong First Medical University &amp; Shandong Academy of Medical Sciences  Jinan China"},{"name":"School of Pharmacy Jining Medical University  Rizhao China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4269-5712","authenticated-orcid":false,"given":"Zhen","family":"Zhang","sequence":"additional","affiliation":[{"name":"School of Pharmacy Jining Medical University  Rizhao China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,12,30]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.talanta.2023.124721"},{"issue":"12","key":"e_1_2_10_3_1","first-page":"156","article-title":"Novel Matrixes for Mass Spectrometry Imaging and Research Progress of It in Analyzing Biological Samples","volume":"38","author":"Miao Y. 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