Cheese, a product of microbial fermentation, may be defined as a protein matrix entrapping fat, moisture, minerals and solutes as well as dispersed bacterial colonies. The growth and physiology of bacterial cells in these colonies may be influenced by the microenvironment around the colony, or alternatively the cells within the colony may modify the microenvironment (e.g., pH, redox potential) due to their metabolic activity. To date there remains a significant knowledge gap relating to the degree of micro-heterogeneity in compositional and biochemical parameters such as pH within the cheese matrix and its relationship with microbial, enzymatic and physiochemical parameters and ultimately with cheese quality, consistency and ripening patterns. The objective of this study was to investigate whether pH micro-heterogeneity exists in cheese matrices. For the first time and arising from the development of a method using fluorescent lifetime imaging (FLIM) microscopy with Orgeon green 488 dye, it is now possible to examine cheese for localized differences in pH. A ripened, semi-hard, dry salted cheese manufactured with thermophillic cultures was analyzed. Our study showed micro-heterogeneity in pH within the matrix of that cheese with pH ranging between 4.0 and 5.5. This is particularly interesting as it shows, contrary to previous assumptions, that the pH of a cheese matrix is not homogenous at micro-scale but contains localized variation. This may be due to localized differences in the aqueous phase or concentrations of constituents of the aqueous phase including lactose, lactate, minerals or salt. It may also be influenced by variations in buffering capacity of the surrounding cheese matrix. It is envisaged that future work using this method will focus on determining whether manufacture processes influence pH at local level within different cheese matrices and whether different cheese types may have different patterns of micro heterogeneity. It is also envisaged that this methodology will be employed to examine a broad range of food matrices in differing food products.