Bacteriophages are promising bactericidal agents for controlling growth of pathogenic and spoilage bacteria in fresh produce and in minimally-processed food. However, there is a lack of quantitative understanding of phage-bacteria interactions within a food matrix. To address this unmet need, the interactions of lytic bacteriophages with target model bacteria on the surface and interior (infiltrated) of a fresh produce were characterized using bioluminescence spectroscopy and imaging approaches. The results showed that the phages applied to the surface of lettuce leaves effectively reduced the level of bacterial targets that were present both on the surface and inside of lettuce leaves. However, there were significant differences in the initial rate of decrease of bacteria in the three model systems examined in this study: (a) homogenous well-mixed solution, (b) inoculated on the surface, and (c) infiltrated into the interior of lettuce leaves. The results also demonstrate the application of bioluminescence imaging for non-invasive measurement of interactions between bactericidal agents and target pathogens over an extended period of time in lettuce samples. The number of bacterial cells in lettuce samples before and after phage treatment predicted based on bioluminescence measurements was in agreement with the number calculated by conventional plate counting methods. In summary, the results highlight a non-invasive real-time imaging approach to quantitatively measure interactions of bacteriophages with both the surface inoculated and the infiltrated model bacterial cells in leafy greens. (c) 2012 Elsevier Ltd. All rights reserved.