| Summary: | Contamination of fresh cucumbers (<i>Cucumis sativus</i> L.) with <i>Escherichia coli</i> O157:H7 can impact the health of consumers. Despite this, the pertinent mechanisms underlying <i>E. coli</i> O157:H7 colonization and internalization remain poorly documented. Herein we aimed to elucidate these mechanisms in cucumbers using an inverted fluorescence microscope and hyperspectral microscopy. We observed that <i>E. coli</i> O157:H7 primarily colonized around the stomata on cucumber epidermis without invading the internal tissues of intact cucumbers. Once the bacterial cells had infiltrated into the internal tissues, they colonized the cucumber placenta and vascular bundles (xylem vessels, in particular), and also migrated along the xylem vessels. Moreover, the movement rate of <i>E. coli</i> O157:H7 from the stalk to the flower bud was faster than that from the flower bud to the stalk. We then used hyperspectral microscope imaging to categorize the infiltrated and uninfiltrated areas with high accuracy using the spectral angle mapper (SAM) classification method, which confirmed the results obtained upon using the inverted fluorescence microscope. We believe that our results are pivotal for developing science-based food safety practices, interventions for controlling <i>E. coli</i> O157:H7 internalization, and new methods for detecting <i>E. coli</i> O157:H7-plant interactions.
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