Microbial pathogen infiltration in fresh leafy greens is a significant food safety risk factor. In various postharvest operations, vacuum cooling is a critical process for maintaining the quality of fresh produce. The overall goal of this study was to evaluate the risk of vacuum cooling-induced infiltration of Escherichia coli O157:H7 into lettuce using multiphoton microscopy. Multiphoton imaging was chosen as the method to locate E. coli O157:H7 within an intact lettuce leaf due to its high spatial resolution, low background fluorescence, and near-infrared (NIR) excitation source compared to those of conventional confocal microscopy. The variables vacuum cooling, surface moisture, and leaf side were evaluated in a three-way factorial study with E. coli O157:H7 on lettuce. A total of 188 image stacks were collected. The images were analyzed for E. coli O157:H7 association with stomata and E. coli O157:H7 infiltration. The quantitative imaging data were statistically analyzed using analysis of variance (ANOVA). The results indicate that the low-moisture condition led to an increased risk of microbial association with stomata (P < 0.05). Additionally, the interaction between vacuum cooling levels and moisture levels led to an increased risk of infiltration (P < 0.05). This study also demonstrates the potential of multiphoton imaging for improving sensitivity and resolution of imaging-based measurements of microbial interactions with intact leaf structures, including infiltration. V acuum cooling is a postharvest-processing operation that preserves the quality of fresh produce by rapidly reducing its temperature. Cooling fresh produce to refrigerated temperatures increases shelf life and product quality by reducing cellular activity (1). The vacuum cooling process cools fresh produce by lowering pressure within a sealed chamber and inducing surface moisture evaporation (2-5). Evaporating the surface moisture reduces the enthalpy in the system by taking energy from the surrounding tissue, thereby reducing the temperature in fresh produce. The product may be sprayed with water during the vacuum cooling process in order to prevent excessive water loss from surface evaporation (4, 5).A recent study suggested that vacuum cooling may increase the risk of microbial infiltration into fresh produce, thereby increasing the risk of food-borne illness from microbial food pathogens (6). Microbial infiltration of pathogens in fresh leafy greens significantly increases the risk of food-borne illness, as the infiltrated microbes cannot be effectively removed or treated utilizing standard sanitation and washing processes (6, 7). Microbial infiltration has been reported in both preharvest and postharvest processing of leafy greens (7,8). During postharvest, microbial infiltration mechanisms are predominantly associated with stomata in intact leaf tissue and in some cases associated with damage to the leaf tissue due to the presence of nutrients from these openings in the plant tissue (6-9). Thus, the reported increase in microbial i...