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Studies of the location and distribution of biofilms in tonsil specimens have thus far been limited to either high\u2010magnification methods such as electron microscopy, which enables high\u2010resolution imaging but only from a tiny tissue volume, or lower magnification techniques such as light microscopy, which allow imaging of larger specimens but with poor spatial resolution. To overcome these limitations, we report the use of multimodal optical mesoscopy to visualise and quantify the number and spatial distribution of Gram\u2010positive biofilms in fresh, excised paediatric tonsils. This methodology supports simultaneous imaging of both the tonsil host and biofilms in whole mounts of tissue up to 5\u00a0mm \u00d7 5\u00a0mm \u00d7 3\u00a0mm with subcellular resolution throughout. A quantitative assessment of 36 tonsil specimens revealed no statistically significant difference between biofilm presence on the tonsil surface and the interior of the tonsil. 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