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However, traditional nucleic acid amplification in aqueous solutions tends to lose spatial information because of the high mobility of molecules. Similar to a cellular matrix, hydrogels with biomimetic 3D nanoconfined spaces can limit the free diffusion of nucleic acids, thereby allowing for ultrafast in situ enzymatic reactions. In this study, hydrogel\u2010based in situ space\u2010confined interfacial amplification (iSCIA) is developed for direct imaging of single nucleic acid and single pathogen on biological samples without formaldehyde fixation. With a polyethylene glycol hydrogel coating, nucleic acids on the sample are nanoconfined with restricted movement, while in situ amplification can be successfully performed. As a result, the nucleic acids are lighted\u2010up on the large\u2010scale surface in 20\u00a0min, with a detection limit as low as 1 copy\/10 cm<jats:sup>2<\/jats:sup>. Multiplex imaging with a deep learning model is also established to automatically analyze multiple targets. Furthermore, the iSCIA imaging of pathogens on plant leaves and food is successfully used to monitor plant health and food safety. The proposed technique, a rapid and flexible system for in situ imaging, has great potential for food, environmental, and clinical applications.<\/jats:p>","DOI":"10.1002\/advs.202407055","type":"journal-article","created":{"date-parts":[[2024,10,7]],"date-time":"2024-10-07T10:54:56Z","timestamp":1728298496000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["Space\u2010Confined Amplification for In Situ Imaging of Single Nucleic Acid and Single Pathogen on Biological Samples"],"prefix":"10.1002","volume":"11","author":[{"given":"Tao","family":"Yang","sequence":"first","affiliation":[{"name":"College of Biosystems Engineering and Food Science Zhejiang University  Hangzhou 310058 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dong","family":"Li","sequence":"additional","affiliation":[{"name":"College of Biosystems Engineering and Food Science Zhejiang University  Hangzhou 310058 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