Progress in molecular imaging of tissue-engineered constructs is currently limited by the poor realisation of sensor biocompatibility (design, delivery, toxicity and performance within the biological material) and the lack of standardised well-accepted assay procedures. I will address these issues by capitalizing on my group’s recent pioneering work in luminescence lifetime imaging microscopy of tissue engineering of adult stem cell-derived organoids. My project will focus on design of biosensor scaffolds, their extensive evaluation in multi-parameter fluorescence (FLIM) and phosphorescence (PLIM) microscopy, consolidation and optimisation of the imaging workflow. Collectively, this methodology will enable assessing extracellular, cellular, luminal and microbiota-derived cues (oxygenation, redox, acidification, mitochondrial function, mapping of distinct subpopulations of live proliferating cells) in the biofilms, intestinal organoids (‘mini-gut’) and other ex vivo / in vivo models employed in microbiology, tissue engineering and regenerative medicine. The novel ‘biosensor toolkit’ will be immediately engaged in bio-printing and physiological studies of stem cell niche metabolism in intestinal organoids such as precise live mapping and modelling O2, pH, calcium and glucose gradients, targeting the effects of vitamin D3 deficiency on the stem cell niche ageing, and others. I expect that designed materials will also provide a framework for production of active remodelling of extracellular matrices, in order to engineer the scaffolds with dynamic growth characteristics, subsequent supra-organoid tissue constructs and bio-fabricated tissues. It is anticipated that produced biosensors, imaging approaches and materials will be highly useful to the broad community of tissue engineering and bio-fabrication specialists by advancing imaging in cancer, biofilms and other areas of the life sciences.