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Techniques that dynamically assess the maturation of tissue engineered constructs allow more efficient longitudinal control of developmental parameters than traditional destructive analyses, enhancing the likelihood of successful outcomes. We present a non-destructive and minimally invasive imaging method to monitor the growth of engineered vascular tissue based on label-free fluorescence lifetime imaging (FLIm) using a single fiber optic interface. We demonstrate the potential of the fiber-based FLIm system on vascular grafts composed of antigen removed bovine pericardium extracellular scaffolds seeded with human endothelial or mesenchymal stem cells. Tissue constructs are illuminated with 355 nm pulsed laser light that excites tissue autofluorescence, stemming from scaffold proteins (e.g., collagen), and cellular metabolic co-factors (i.e., NADH and FAD). Fluorescence lifetime images are acquired by scanning the distal tip of a multimode fiber across the sample surface, to deliver fluorescence excitation and collect fluorescence emission. A wavelength selection module is used to spectrally separate autofluorescence into four spectral bands that were selected to match the emission peaks of the main tissue fluorophores. By examining the relative intensity and mean fluorescence lifetime in each spectral band we identify the composition of engineered tissues, and evaluate the progression of recellularization. The fiber-based apparatus is compatible with imaging a range of sample geometries including planar and tubular constructs, and imaging in regions of restricted space such as inside tissue bioreactors, or in vivo. Future applications for the system include longitudinal monitoring of the luminal surface of engineered vascular tissues, or intravascular imaging in vivo to monitor viability of vascular implants.
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