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Quantitative Multi‐Parametric Microscopy Analysis of Live 3D Cell Models

Three‐dimensional (3D) cell models are at the forefront of cancer cell biology and tissue engineering, providing opportunity for better in vitro mimicking of cell morphology, membrane transport, physiological O2, pH and metabolite diffusion gradients, lacked in conventional cell culture. However, studying the biochemistry of the cell in 3D demands new methods of quantitative fluorescence microscopy such as fluorescence and phosphorescence lifetime imaging microscopies (FLIM, PLIM). Here we report development and application of imaging protocols for quantitative microscopy of molecular oxygen (O2) and temperature (T), combined with analysis of cell death in tumor spheroids from human colon carcinoma HCT116 [1, 2]. By analyzing gradients of pH, O2 and fluorescence‐based viability markers (CellTox Green, TMRM) we optimized the method of spheroid formation, providing best overall cell viability and reproducibility. By measuring T and O2 in spheroid models, we identified T as new metabolic marker, dependent on the 3D organization of cell [2]. We also thoroughly evaluated the available and introduced a number of novel nanoparticle‐based probes for high‐resolution imaging of hypoxia (O2), pH and T by FLIM method. Overall, we see the described methodology as enabling quantitative microscopy of advanced 3D cell models in tissue engineering and regenerative medicine.