Research on cell migration and interactions with the extracellular matrix (ECM) was mostly focused on 2D surfaces in the past. Many recent studies have highlighted differences in migratory behaviour of cells on 2D surfaces compared to complex cell migration modes in 3D environments. When embedded in 3D matrices, cells constantly sense the physicochemical, topological and mechanical properties of the ECM and adjust their behaviour accordingly. Changes in the stiffness of the ECM can have effects on cell morphology, differentiation and behaviour and cells can follow stiffness gradients in a process called durotaxis. Here we introduce a detailed protocol for the assembly of 3D matrices consisting of collagen I/fibronectin and embedding cells for live cell imaging. Further, we will show how the matrix can be stiffened via non-enzymatic glycation and how collagen staining with fluorescent dyes allows simultaneous imaging of both matrix and cells. This approach can be used to image cell migration in 3D microenvironments with varying stiffness, define cell-matrix interactions and the cellular response to changing ECM, and visualize matrix deformation by the cells.