Three-dimensional oxygen concentration monitoring in hydrogels using low-cost phosphorescence lifetime imaging for tissue engineering.

Oxygen concentration measurement in 3D hydrogels is vital in 3D cell culture and tissue engineering. However, standard 3D imaging systems capable of measuring oxygen concentration with adequate precision are based on advanced microscopy platforms, which are not accessible in many laboratories due to the system's complexity and the high price. In this work, we present a fast and low-cost phosphorescence lifetime imaging design for measuring the lifetime of oxygen-quenched phosphorescence emission with 0.25 µs temporal precision and sub-millimeter spatial resolution in 3D. By combining light-sheet illumination and the frequency-domain lifetime measurement using a commercial rolling-shutter CMOS camera in the structure of a conventional optical microscope, this design is highly customizable to accommodate application-specific research needs while also being low-cost as compared to advanced instruments. As a demonstration, we made a fluidic device with a gas-permeable film to create an artificial oxygen gradient in the hydrogel sample. Dye-embedded beads were distributed in the hydrogel to conduct continuous emission lifetime monitoring when nitrogen was pumped through the fluidic channel and changed oxygen distribution in the sample. The dynamics of the changes in lifetime co-related with their location in the gel of size 0.5 mm×1.5 mm×700 µm demonstrate the ability of this design to measure the oxygen concentration stably and precisely in 3D samples.