2.7. Injection time characterisation

A 20 μg mL⁻¹ fluorescein solution (F6377-100G, Sigma-Aldrich) was used to measure injection times and concentrations in the culture chamber of the microfluidic device. Progression of the solution in the chamber was assessed by fluorescence microscopy imaging at 2× magnification. Images were acquired every minute during injection and wash-out of the solution, and every 30 min during the static period. Pixel intensities at different injection times were measured across the centre of the chamber using ImageJ [20]. A calibration curve was established measuring different concentrations of fluorescein in static conditions (no flow). To reduce the effect of photo-bleaching, the microscope epifluorescence lamp was only switched on during image acquisition.