Abstract The tumour microenvironment (TME) strongly influences tumorigenesis and metastasis. Two of the most characterized properties of the TME are acidosis and hypoxia, both of which are considered hallmarks of tumours as well as critical factors in response to anticancer treatments. Currently, various imaging approaches exist to measure acidosis and hypoxia in the TME, including magnetic resonance imaging (MRI), positron emission tomography and optical imaging. In this review, we will focus on the latest fluorescent-based methods for optical sensing of cell metabolism and MRI as diagnostic imaging tools applied both in vitro and in vivo. The primary emphasis will be on describing the current and future uses of systems that can measure intra- and extra-cellular pH and oxygen changes at high spatial and temporal resolution. In addition, the suitability of these approaches for mapping tumour heterogeneity, and assessing response or failure to therapeutics will also be covered.