Melissa Pezzotti

Graduation: [-]

Supervisor: Prof. Francesco S. Pasqualini

Thesis title: Advanced Optical Methods in Tissue Engineering

Abstract: Tissue engineering increasingly relies on live-cell fluorescence imaging to connect cellular behavior with engineered microenvironments and to quantify how cellular state, structure, and function evolve over time. However, scaling live phenotyping toward high-throughput and high-content experiments remains constrained by phototoxicity, limited integration between microfabrication and microscopy workflows, and the difficulty of obtaining quantitative, cell-state-aware readouts in complex three-dimensional models. This thesis addresses these challenges from an engineering perspective by developing modular and dose-conscious tools for interpretable live-cell phenotyping. First, a FUCCIplex-guided adaptive imaging strategy is used to focus confocal acquisition on informative cell-cycle windows, reducing unnecessary illumination during phototoxic mitotic imaging. Second, a vertically integrated workflow combines LIMAP-patterned planar confinement, multiplexed genome-edited readouts, and the Fab2Mic fabrication-to-microscopy correlation pipeline to link microenvironment geometry with structural and cell-cycle behaviors during long-term imaging. Third, TE-DaXi, a custom oblique-plane light-sheet microscope, is introduced as a prototyping platform for fast volumetric bursts and long-term low-dose imaging of tissue-engineering specimens. Finally, a volumetric imaging and analysis workflow is established for hiPSC-derived cardiac organoids, linking cavity architecture with proliferative state through coupled structural-functional readouts and qualitative model-guided interpretation. Together, these contributions connect genetic engineering, microenvironment design, instrument development, and quantitative image analysis toward more controlled, auditable, and context-aware phenotyping workflows for live-cell tissue engineering.