The field of tissue engineering aims to create models that closely mimic the structure of native tissues. This to gain better insights into the cellular and molecular events happening in our tissues. A critical aspect of these models is the cellular microenvironment, which is defined by multiple factors, including the composition of the extracellular matrix (ECM), the presence of soluble signalling molecules, as well as cell-cell and cell-matrix interactions. These elements collectively regulate cellular behavior, influencing processes such as cell differentiation, proliferation, and tissue organization. A comprehensive understanding of the cellular microenvironment is fundamental for the accurate design of both two-dimensional (2D) and three-dimensional (3D) tissue models. As cells are highly responsive to their environment, precise replication and monitoring of these factors are essential for advancing tissue engineering and its applications in regenerative medicine and disease modeling. This thesis investigates factors influencing the cell microenvironment by employing collagen-binding domain-tagged fluorescent biosensors responsive to glucose, lactate, and pH. These biosensors, composed of a ligand-binding domain and a fluorescent reporter protein, undergo fluorescence emission changes upon binding to their factor of interest. Fluorescence lifetime imaging microscopy (FLIM) is employed to quantify these dynamic changes, enabling a detailed metabolic analysis and characterization of 3D tissue models.