Advanced optical methods in tissue engineering

Tissue engineering increasingly depends on live-cell fluorescence imaging to connect cellular behavior with engineered microenvironments and to measure how state, structure, and function change over time. However, scaling phenotyping toward high-throughput and high-content (HT/HC) experiments remains limited by three ongoing challenges: phototoxicity and accumulated photodamage during information-rich acquisition; weak compatibility between microenvironment fabrication and microscopy workflows; and the difficulty of obtaining quantitative, cell-state-aware readouts in complex 3D models under practical constraints of cost, standardization, and imaging stability. This thesis tackles these challenges from an engineering perspective by developing modular, compatible, and dose-controlled tools to improve phenotype interpretability and measurement comparability as experimental platforms, acquisition modes, and analysis pipelines evolve. First, we present a cell biology-guided adaptive imaging strategy to reduce illumination load during highly phototoxic mitotic imaging. By using the multiplexed FUCCIplex cell-cycle (CC) reporter to identify informative time windows, confocal acquisition is focused both in time and space, allowing high-resolution observation of mitotic processes while minimizing unnecessary exposure outside the relevant phase. Second, the thesis advances substrate engineering as a controlled external modulation and develops a vertically integrated workflow for CC-aware phenotyping under planar confinement. Using LIMAP-patterned confinement and multiplexed genome-edited readouts, the developed Fab2Mic fabrication-to-microscopy correlation pipeline translates fabrication coordinates into imaging coordinates, enabling long-term, automated phenotyping that links microenvironment geometry to structural and cell-cycle behaviors within defined planar confinement assays. Third, we introduce TE-DaXi, a custom oblique-plane light-sheet microscope developed as a design and prototyping effort to support volumetric phenotyping in tissue-engineering specimens. TE-DaXi is conceived to combine fast, high-resolution volumetric bursts with long-term, low-dose volumetric imaging for morphology and cell-cycle tracking, providing a platform onto which experimental and analytical workflows can be transferred after further calibration and validation. Lastly, the thesis focuses on a 3D phenotyping workflow using a volumetric imaging system, prioritizing robustness and interpretability to establish an end-to-end pipeline for self-organizing hiPSC-derived cardiac organoids (cardioids). The workflow integrates sample handling, gentle volumetric imaging, and quantitative analysis to link cavity architecture to proliferative state via coupled structural-functional readouts. A two-phase model is adopted as an interpretive layer for observed trends, providing a qualitative framework for comparing cavity morphogenesis regimes across conditions. Together, these contributions connect genetic engineering, microenvironment design, instrument development, and quantitative analysis toward more controlled, auditable, and context-aware phenotyping workflows in live-cell tissue engineering. This thesis provides practical modules and design principles that can support future scaling and cross-platform comparability when validated across additional models, perturbations, and imaging systems.