Light microscopy and fluorescence imaging are two powerful and widely used techniques in bioengineering that can be used to visualize biomolecular interactions and cellular dynamics, providing images of high spatial and temporal resolution. Fluorescence imaging, enhanced by fluorescent dyes, proteins, and nanoparticles, offers high specificity and sensitivity for detecting biomolecules and dynamic biological processes. This chapter gives a comprehensive overview of advances fluorescence imaging modalities such as Fluorescence Lifetime Imaging Microscopy (FLIM), Fluorescence Recovery After Photobleaching (FRAP) and Förster resonance energy transfer (FRET) for their contributions towards overcoming classical imaging barriers in imaging complex biological samples and events. This chapter also features the major applications of advanced light microscopy methods with references in bioengineering ranging from cellular and molecular imaging, tissue engineering, and regenerative medicine to drug discovery, disease diagnosis and prognosis, biomaterials research.