Maestro imaging system

We have previously demonstrated the uptake of TRaMs in MCECs and aortas, and thus here the in vitro and ex vivo uptake of TRaMs was assessed in human bronchial epithelial cells (BEAS-2B) and mouse trachea.¹¹ For in vitro experiments BEAS-2B cells were exposed to simulated cold hyperoxic storage and reperfusion as previously described.^{15 (link)} Cells were exposed to UW or UW augmented with TRaM and following 18 hours of cold storage, were reperfused for 10 and 60 minutes to visualize TRaM internalization using Olympus Fluoview FV10i LIV Confocal Microscope (Olympus, NC). Donor tracheas were harvested for analysis of ex vivo delivery of NPs. Tissue segments were stored on ice for 6 hours in UW solution, or UW solution augmented with increasing doses of RaM and TRaM (100, 500, and 1000 ng mL⁻¹). Fluorescence multispectral imaging was performed using the Maestro Imaging System (PerkinElmer, MA). Multispectral images were acquired under a constant exposure of 2000 ms with an orange filter acquisition setting of 630–850 nm in 2 nm increments. Multispectral images were unmixed into their component spectra (Dylight 680, autofluorescence, and background) and these component images were used to gain quantitative information in terms of average fluorescence intensity by creating regions of interest (ROIs) around the organs in the Dylight 680 component images.